Engineered immunoglobulin fc polypeptides

ABSTRACT

Methods and compositions involving polypeptides having an aglycosylated antibody Fc domain. In certain embodiments, polypeptides have an aglycosylated Fc domain that contains one or more substitutions compared to a native Fc domain. Additionally, some embodiments involve an Fc domain that is binds some Fc receptors but not others. For example, polypeptides are provided with an aglycosylated Fc domain that selectively binds FcγRIIa, but that is significantly reduced for binding to the highly homologous FcγRIIb receptors. Furthermore, methods and compositions are provided for promoting antibody-dependent cell-mediated toxicity (ADCC) using a polypeptide having a modified aglycosylated Fc domain and a second non-Fc binding domain, which can be an antigen binding region of an antibody or a non-antigen binding region. Some embodiments concern antibodies with such polypeptides, which may have the same or different non-Fc binding domain.

This application claims priority to U.S. provisional patent application61/440,297 filed on Feb. 7, 2011, which is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of proteinengineering. More particularly, it concerns improved methods andcompositions for the screening of combinatorial antibody Fc librariesexpressed in bacteria.

2. Description of Related Art

Currently recombinant therapeutic antibodies have sales of well over $10bn/yr and with a forecast of annual growth rate of 20.9%, they areprojected to increase to $25 bn/yr by 2010. Monoclonal antibodies (mAbs)comprise the majority of recombinant proteins currently in the clinic,with more than 150 products in studies sponsored by companies locatedworldwide (Pavlou and Belsey, 2005). In terms of therapeutic focus, themAb market is heavily focused on oncology and arthritis, immune andinflammatory disorders, and products within these therapeutic areas areset to continue to be the key growth drivers over the forecast period.As a group, genetically engineered mAbs generally have higherprobability of FDA approval success than small-molecule drugs. At least50 biotechnology companies and all the major pharmaceutical companieshave active antibody discovery programs in place.

The original method for isolation and production of mAbs was firstreported at 1975 by Milstein and Kohler (Kohler and Milstein, 1975), andit involved the fusion of mouse lymphocyte and myeloma cells, yieldingmouse hybridomas. Therapeutic murine mAbs entered clinical study in theearly 1980s; however, problems with lack of efficacy and rapid clearancedue to patients' production of human anti-mouse antibodies (HAMA) becameapparent. These issues, as well as the time and cost consuming relatedto the technology became driving forces for the evolution of mAbproduction technology. Polymerase Chain Reaction (PCR) facilitated thecloning of monoclonal antibodies genes directly from lymphocytes ofimmunized animals and the expression of combinatorial library offragments antibodies in bacteria (Orlandi et al., 1989). Later librarieswere created entirely by in vitro cloning techniques using naïve geneswith rearranged complementarity determining region 3 (CDR3) (Griffithsand Duncan, 1998; Hoogenboom et al., 1998). As a result, the isolationof antibody fragments with the desired specificity was no longerdependent on the immunogenicity of the corresponding antigen. Moreover,the range of antigen specificities in synthetic combinatorial librarieswas greater than that found in a panel of hybridomas generated from animmunized mouse. These advantages have facilitated the development ofantibody fragments to a number of unique antigens including smallmolecular compounds (haptens) (Hoogenboom and Winter, 1992), molecularcomplexes (Chames et al., 2000), unstable compounds (Kjaer et al., 1998)and cell surface proteins (Desai et al., 1998).

In microbial cells, display screening may be carried out by flowcytometry. In particular, Anchored Periplasmic Expression (APEx) isbased on anchoring the antibody fragment on the periplasmic face of theinner membrane of E. coli followed by disruption of the outer membrane,incubation with fluorescently labeled target and sorting of thespheroplasts (U.S. Pat. No. 7,094,571). APEx was used for the affinitymaturation of antibody fragments (Harvey et al., 2004; Harvey et al.,2006). In one study over 200-fold affinity improvement was obtainedafter only two rounds of screening.

One important mechanism underlying the potency of antibody therapeuticsis the ability of antibody to recruit immune cells to a target antigen(or cell). Thus, the Fc region of an antibody is crucial for recruitmentof immunological cells and antibody dependent cytotoxicity (ADCC). Inparticular, the nature of the ADCC response elicited by antibodiesdepends on the interaction of the Fc region with receptors (FcRs)located on the surface of many cell types. Humans contain five differentclasses of Fc receptors. In addition haplotypes, or genetic variants ofdifferent FcRs belonging to a particular class are known. The binding ofan antibody to FcRs determines its ability to recruit otherimmunological cells and the type of cell recruited. Hence, the abilityto engineer antibodies that can recruit only certain kinds of cells canbe critically important for therapy.

However, to the inventors' knowledge, previous attempts to engineer Fcdomains have been performed using mammalian-expressed IgG molecules.Mammalian antibodies are glycosylated. The carbohydrate chain isattached to the Fc region and alters the conformation of the protein andenables the antibody to bind to FcRs. In contrast, aglycosylatedantibodies produced in bacteria cannot bind to FcRs and therefore areunable to elicit ADCC. It is desirable to engineer aglycosylatedantibodies that are capable of eliciting ADCC and thus benefit from thelower production costs that are derived from bacterial expression.

Second, and most importantly, mammalian antibodies with engineered Fcregions display increased binding to a particular FcR of interest but inaddition they are still capable of binding to other FcRs with normalaffinity. Thus, while such antibodies are more selective than themolecules naturally produced by the immune system they can nonethelessstill mediate undesirable immunological responses.

Nonetheless, all high throughput antibody screening technologiesavailable to-date rely on microbial expression of antibody fragments.The use of antibody fragments rather than intact or full length IgGs, inthe construction and screening of libraries has been dictated bylimitations related to the expression of the much larger IgGs inmicroorganisms. IgG libraries have never before been expressed orscreened using microorganisms such as bacteria or yeasts. As a resultthe isolation of antigen binding proteins has been carried outexclusively using antibody fragments that are smaller and much easier toproduce. Once isolated, such antibody fragments have to then be fused tovectors that express full length immunoglobulins which in turn areexpressed preferentially in mammalian cells such as CHO cells.

E. coli possesses a reducing cytoplasm that is unsuitable for thefolding of proteins with disulfide bonds which accumulate in an unfoldedor incorrectly folded state (Baneyx and Mujacic, 2004). In contrast tothe cytoplasm, the periplasm of E. coli is maintained in an oxidizedstate that allows the formation of protein disulfide bonds. Notably,periplasmic expression has been employed successfully for the expressionof antibody fragments such as Fvs, scFvs, Fabs or F(ab′)2s (Kipriyanovand Little, 1999). These fragments can be made relatively quickly inlarge quantities with the retention of antigen binding activity.However, because antibody fragments lack the Fc domain, they do not bindthe FcRn receptor and are cleared quickly; thus, they are onlyoccasionally suitable as therapeutic proteins (Knight et al., 1995).Until recently, full-length antibodies could only be expressed in E.coli as insoluble aggregates and then refolded in vitro (Boss et al.,1984; Cabilly et al., 1984). Clearly this approach is not amenable tothe high throughput screening of antibody libraries since with thecurrent technology it is not possible to refold millions or tens ofmillions of antibodies individually. A further problem is that since E.coli expressed antibodies are not glycosylated, they fail to bind tocomplement factor 1q (C1q) or Fc and many other Fc receptors. However,aglycosylated Fc domains can bind to the neonatal Fc receptorefficiently (FcRn). Consequently bacterially expressed aglycosylatedantibodies do exhibit serum persistence and pharmacokinetics similar tothose of fully glycosylated IgGs produced in human cells. Nonetheless,since the aglycosylated antibodies fail to elicit complement activationand can not mediate the recruitment of immune cells such as macrophages,they have previously been ineffective for many therapeutic applications.

In humans there are five major FcγRs. IgG antibodies bind to all thesereceptors with varying affinities. Of note, out of the 5 FcγRs, fourinduce activating or pro-inflammatory responses, while one FcγRIIbinduces anti-inflammatory or inhibitory responses. All naturallyproduced antibodies and also recombinant glycosylated antibodiesproduced by tissue culture contain Fc domains that bind to both theactivating and the inhibitory FcγRs. The ability of antibodies to induceactivating ADCC depends on the ratio of binding affinities to theactivating FcγRs vs the inhibitory FcγRIIb (A/I ratio) (Boruchov et al.2005; Kalergis et al., 2002). Efforts to enhance the A/I ratio byengineering mutations in glycosylated antibodies that increase bindingto activating FcγRs and reduce binding to FcγRIIb have been met withlittle success to a large part because the latter is 96% homologous tothe activating FcγRs. Different FcγR effector functions include(antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine release,phagocytosis, and maturation. Fc domains engineered to have selectiveeffector functions could provide physiological benefits.

SUMMARY OF THE INVENTION

This disclosure provides compounds and methods involving aglycosylatedantibody Fc domains that bind to Fc receptors.

In some embodiments, there are compositions involving a polypeptide thathas an aglycosylated Fc domain from an antibody (“antibody Fc domain”).In additional embodiments, the aglycosylated Fc domain is a variant of awild-type Fc domain such that the variation allows the Fc domain tospecifically bind to one or more Fc receptors. In some embodiments, apolypeptide with an aglycosylated Fc domain variant is able to bind onlya subset of Fc receptors that a polypeptide with glycosylated version ofthe wild-type Fc domain (“glycosylated wild-type Fc domain”) can bind.In specific embodiments, the polypeptide with an aglycosylated Fc domainvariant can specifically bind FcγRI; in some cases, it has the affinityor binding ability that is within 2-fold of a polypeptide having aglycosylated wild-type Fc domain. In other embodiments, additionally oralternatively, the polypeptide with an aglycosylated Fc domain varianthas significantly reduced affinity or binding ability (50-fold orgreater reduction) compared to a polypeptide having a glycosylatedwild-type Fc domain. In certain embodiments, the polypeptide with anaglycosylated Fc domain variant has a significantly reduced affinity toor ability to bind FcγRIIb relative to the affinity towards itshomologous activating receptor Fc□RIIa. It is contemplated that apolypeptide may have an affinity or binding ability for FcγRI that iscomparable (within 2-fold), as well as significantly reduced affinity orbinding ability for FcγRIIB, both as compared to a polypeptide having aglycosylated wild-type Fc domain.

As used herein, the term “affinity” refers to the equilibrium constantfor the reversible binding of two agents and is expressed as Kd.Affinity of a binding domain to its target can be, for example, fromabout 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1picomolar (pM), or from about 100 nM to about 1 femtomolar (fM);alternatively, it can be between 100 nM and 1 nM or between 0.1 nM and10 nM. Moreover, it is contemplated that agents specifically bind whenthere is an affinity between the two agents that is in the affinityranges discussed above.

An antibody Fc domain may be the Fc domain of an IgA, IgM, IgE, IgD orIgG antibody or a variant thereof. In certain embodiments, the domain isan IgG antibody Fc domain such as an IgG1, IgG2a, IgG2b, IgG3 or IgG4antibody Fc domain. Furthermore, the antibody Fc domain may be definedas a human Fc domain, in which case it specifically binds one or morehuman Fc receptors. In certain aspects, the Fc domain may be an IgG1 Fcdomain, such as the Fc domain of an anti-HER2 antibody, morespecifically, the Fc domain of trastuzumab. It is contemplated that insome embodiments an entire polypeptide is aglycosylated or that in otherembodiments only a portion of the polypeptide is aglycosylated, such asthe Fe domain. It is also contemplated that a polypeptide may containone or more regions from an antibody in addition to the Fc domain. Apolypeptide may contain an antigen binding domain from an antibody.Moreover, multiple polypeptides may form an antibody or antibody-likeprotein.

In some embodiments, there is a polypeptide comprising an aglycosylatedantibody Fc domain capable of binding a human FcR polypeptide, whereinthe Fc domain comprises particular amino acid substitutions. In someembodiments there are multiple amino acid substitutions. Withsubstitutions in the human aglycosylated Fc domain, embodiments includea polypeptide with a human Fc domain having an amino acid substitutionat amino acids 298 and 299 and at least one additional substitution atthe following position or positions: 382; 382 and 263; 382, 390 and 428;392, 382, 397 and 428; 315, 382 and 428 or 268, 294, 361, 382 and 428.

In some cases it is contemplated that the substitution at amino acid 298is glycine (S298G) and the substitution at amino acid 299 is alanine(T299A).

Where the additional amino acid substitution is at amino acid 382, apreferred substitution is valine (E382V).

Where the additional substitution is at amino acids 382 and 263, inpreferred embodiments, the substitution at amino acid 382 is valine(E382V) and the substitution at amino acid 263 is glutamic acid (V263E).

Where the additional substitution is at amino acids 382, 390 and 428, inpreferred embodiments, the substitution at amino acid 382 is valine(E382V), the substitution at amino acid 390 is aspartic acid (N390D) andthe substitution at amino acid 428 is leucine (M428L).

Where the additional amino acid substitution is at amino acids 392, 382,397 and 428, inn preferred embodiments, the substitution at amino acid382 is valine (E382V), the substitution at amino acid 392 is glutamicacid (K392E), the substitution at amino acid 397 is methionine (V392M)and the substitution at amino acid 428 is leucine (M428L).

Where the additional amino acid substitution is at amino acids 315, 382and 428, in preferred embodiments, the substitution at amino acid 315 isaspartic acid (N315D), the substitution at amino acid 382 is valine(E382V), and the substitution at amino acid 428 is leucine (M428L).

Where the additional substitution is at amino acids 268, 294, 361, 382and 428, in preferred embodiments, the substitution at amino acid 268 isproline (H268P), the substitution at position 294 is lysine (E294K), thesubstitution at amino acid 361 is serine (N361S), the substitution atamino acid 382 is valine (E382V) and the substitution at amino acid 428is leucine (M428L).

In some embodiments, a polypeptide has an aglycosylated human Fc domainwith a substitution in amino acids 382 and 428 and also has at least oneadditional substitution in the upper CH2 region.

Embodiments involve a polypeptide having an aglycosylated Fc domain thatis capable of specifically binding one or more particular human FcRpolypeptides. In some embodiments, the aglycosylated Fc domain has beenmutated so that it can bind one or more of FcγRIa, FcγRIIa, FcγRIIb,FcγRIIc, FcγRIIIa, FcγRIIIb, or FcαRI. It is contemplated that thebinding to one or more of these particular human FcR polypeptides iswithin 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (or any rangederivable therein) of the binding seen with a glycosylated Fc region orthat the binding is altered (increased or decreased) by at least or atmost 50, 60, 70, 80, 90, or 100% (or any range derivable therein)relative to a wild-type glycosylated Fc domain. Alternatively, relativebinding capabilities between polypeptides having a mutated andaglycosylated Fc domain and polypeptides having a glycosylated andwild-type Fc domain may be expressed in terms of X-fold differences(increased or decreased). For example, there may be at least or at mostat least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold difference, or anyrange derivable therein).

In some embodiments, a polypeptide with a mutated aglycosylated Fcdomain is capable of specifically binding an FcγRI polypeptide. In somecases, it binds at a level within 2-fold of the level of binding by apolypeptide having a glycosylated and wild-type Fc domain. In otherembodiments, the level of binding is within at least 2-, 3-, 4-, 5-, 6-,7-, 8-, 9-, or 10-fold a glycosylated and wild-type Fc domain. Forexample, the K_(D) value for a particular Fc receptor and either apolypeptide with the aglycosylated Fc domain variant or a polypeptidewith a glycosylated and wild-type Fc domain is within at least 2- or3-fold in embodiments described herein. In some embodiments, apolypeptide has at least a 2-fold reduction in pH-dependent FcRn bindingcompared to polypeptide with an aglycosylated wild-type antibody Fcdomain. In additional embodiments,

Polypeptides described herein may include a linker in some embodiments.In further embodiments, the linker is a conjugatable linker. In someembodiments, the polypeptide contains an Fc domain from an antibody. Itmay contain other regions from an antibody, such as another bindingdomain. The additional binding domain is not an FcR binding domain incertain embodiments. In some embodiments, it may contain an antigenbinding site or domain from an antibody. This would include all or partof the variable region from an antibody. In other embodiments, apolypeptide contains an Fc domain from an antibody but another bindingdomain that is a non-FcR binding domain. In some embodiments, the non-Fcbinding region is not an antigen binding site of an antibody butspecifically binds a cell-surface protein. In some cases, a cell-surfaceprotein that the non-Fc binding region recognizes is a receptor. In someembodiments, a cell-surface receptor is a tyrosine kinase. In additionalembodiments, a polypeptide has a non-Fc binding region capable ofbinding multiple tyrosine kinase receptors. In some embodiments, such anon-Fc binding region is capable of binding one or more of VEGFreceptors, PDGF receptors, EGFR receptors, ErbB-2 receptors, EGFreceptors, HGF receptors, and other Src-like tyrosine kinase receptors,or a combination thereof. It is also specifically contemplated thatpolypeptides have an antigen binding region that recognizes one or moreof these receptor tyrosine kinases.

Other polypeptides include those having an aglycosylated Fc domaincapable of binding an FcRγI polypeptide and a second binding domain,wherein the second binding domain is capable of specifically binding acell-surface molecule. In some embodiments, the second binding domain isan antigen binding domain of an antibody (“antibody antigen bindingdomain”). In some cases, the second binding domain is not an antibodyantigen binding domain. In some embodiments, the second binding domainis capable of specifically binding a cell-surface molecule that is aproteinaceous molecule. The second binding domain may be a ligand for acell-surface receptor or it may be a receptor for a cell-surface ligand.

Embodiments also concern a nucleic acid that encodes any of thepolypeptides discussed herein. The nucleic acid may be isolated and/orrecombinant. It may be a nucleic acid segment that is isolated and/orrecombinant. In some embodiments, the nucleic acid is DNA while inothers it is RNA. In certain embodiments, the nucleic acid is a DNAsegment. In other embodiments, the nucleic acid is an expression vectorthat is capable of expressing any of the polypeptides having an Fcbinding domain with one or more substitutions that specifically binds ahuman FcR polypeptide. A nucleic acid may encode one or morepolypeptides discussed above, which, depending on how the polypeptide isproduced may or may not be glycosylated.

In some embodiments, there are nucleic acids encoding a polypeptide withan Fc domain capable of specifically binding a human FcR polypeptide.The nucleic acid may be placed in a host cell that can express thepolypeptide, particularly an aglycosylated version of the polypeptide.The host cell may be a prokaryotic cell, such as a bacterial cell.Alternatively, the host cell may be an eukaryotic cell, such as amammalian cell. In some embodiments, a host cell contains a firstexpression vector, though it may comprises a second expression vector aswell. Because some antibodies are made of multiple polypeptides, a hostcell that expresses these polypeptides is contemplated in someembodiments. For example, in some embodiments there is a host cell thatincludes a second expression vector that encodes a polypeptidecomprising an immunoglobulin light chain.

In some embodiments, there is a population of host cells, wherein thepopulation contains a plurality of host cells that express polypeptideshaving different Fc domains. It is contemplated that the amino acidsequence of any two different Fc domains differs in identity by lessthan 20%, 15%, 10%, 5% or less.

In some embodiments there are methods of making the polypeptidesdescribed herein (polypeptides having an aglycosylated Fc region) aswell as methods of using these polypeptides. Any of these methods may beimplemented with respect to any of the polypeptides described herein.

In some embodiments there are methods for preparing an aglycosylatedpolypeptide comprising: a) obtaining a host cell capable of expressingan aglycosylated antibody comprising an Fc domain capable of binding anFcR polypeptide, wherein the Fc domain comprises an above-mentionedsubstitution, that is, an amino acid substitution at amino acids 298 and299 and at least one additional substitution at the following positionor positions: 382; 382 and 263; 382, 390 and 428; 392, 382, 397 and 428;315, 382 and 428 or 268, 294, 361, 382 and 428; b) incubating the hostcell in culture under conditions to promote expression of theaglycosylated antibody; and, c) purifying expressed antibody from thehost cell. In some embodiments, the host cell is a prokaryotic cell,such as a bacterial cell. In further embodiments, methods involvecollecting expressed antibody from the supernatant, which may be doneprior to purification.

In some embodiments methods involve purifying the antibody from thesupernatant. This may involve subjecting the antibodies from thesupernatant to filtration, HPLC, anion or cation exchange, highperformance liquid chromatography (HPLC), affinity chromatography or acombination thereof. In some embodiments, methods involve affinitychromatography using staphylococcal Protein A, which binds the IgG Fcregion. Other purification methods are well known to those of ordinaryskill in the art.

In some embodiments, the aglycosylated polypeptide or antibody iscapable of specifically binding an activating FcR polypeptide, whichrefers to an FcR polypeptide that activates one or more immune cells.Activating polypeptides include FcγRI, IIa, IIIa, IIb, and IIIc. FcγRIIbis an inhibitory FcR polypeptide. In further embodiments, theaglycosylated polypeptide or antibody no longer binds an inhibitory FcRpolypeptide at a level comparable to a glycosylated, wild-type Fcdomain. In specific embodiments, an aglycosylated polypeptide orantibody specifically binds an FcγRI polypeptide. In furtherembodiments, the aglycosylated polypeptide or antibody has a reducedcapability to bind an FcγRIIb polypeptide, wherein its affinity is atleast 50-fold less than a glycosylated, wild-type version of thepolypeptide or antibody. In certain embodiments, the aglycosylatedantibody is an aglycosylated version of a therapeutic antibody, whichrefers to an antibody used in therapy or treatment for a disease orcondition. Any antibody or polypeptide discussed herein, including thosediscussed above, may be used in implementing methods for inducing animmune response. An example of a therapeutic antibody is trastuzumab.

In some embodiments, methods involve bacterial cells that are E. colicells. In additional embodiments, the Fc domain is an IgG, IgA or IgE Fcdomain. In further embodiments, the population of Gram negativebacterial cells comprise a plurality of nucleic acids encoding theplurality of aglycosylated Fc domains. In some cases the plurality ofnucleic acids further encodes a membrane secretion signal fused to theplurality of aglycosylated Fc domains. A membrane secretion signal maybe PelB or DsbA. Additionally, the aglycosylated Fc domain may include ahinge, CH2 and CH3 region. In certain embodiments, the aglycosylatedpolypeptide comprises an eukaryotic FcR domain. In some embodiments,there is a polypeptide with an Fc domain that specifically binds one ofthe polypeptides of Table 1. In certain embodiments, the Fc domain bindshuman FcγRIa, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, FcαRI orC1q. In other embodiments, it has reduced binding affinity for FcγRIIbrelative to a glycosylated and wild-type version of the Fc domain.Specific methods are disclosed in WO 2008/137475, which is herebyincorporated by reference.

Other embodiments involve methods for optimizing Fc binding to one ormore specific FcR polypeptides of an aglycosylated polypeptide having anFc domain comprising: a) obtaining a population of Gram negativebacterial cells, cells of which population express a aglycosylatedpolypeptide comprising an Fc domain in their periplasm, wherein thepopulation expresses a plurality of different polypeptides expressingdifferent mutated Fc domains; b) contacting the bacterial cells with afirst FcR polypeptide under conditions to allow contact between the FcRpolypeptide and the aglycosylated Fc domains, wherein the FcRpolypeptide is FcγRIa, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, orFcαRI; and c) selecting at least one bacterial cell based on binding ofthe aglycosylated Fc domain to the first FcR polypeptide. Any of theembodiments discuss above may apply to the implementation of thesemethods.

Embodiments discussed in the context of a methods and/or composition ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

As used herein the terms “encode” or “encoding” with reference to anucleic acid are used to make the invention readily understandable bythe skilled artisan however these terms may be used interchangeably with“comprise” or “comprising” respectively.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. SDS-PAGE gel showing the purified Fc fragments, wild type Fc andFc2a proteins. Lane M: molecular weight standards; lane 1: Wild type Fc;lane 2; Fc2a (S298G/T299A).

FIG. 2. ELISA assays showing the affinity of aglycosylated Fc2a (Fcfragments) and aglycosylated trastuzumab-Fc2a (full length IgG) toFcγRIIa. [Note: First bar in each group is RI, second is RIIa and thirdis RIIb)]

FIG. 3. Specificities of aglycosylated trastuzumab Fc variants inbinding to FcγRI, FcγRIIa, and FcγRIIb. [Note: First bar in each groupis RI, second is RIIa and third is RIIb]

FIG. 4. Alignment of X-ray crystal structures of FcγRIIa (PDB: 1FCG) andFcγRIIb (2FCB) showing the high homology between the two proteins.

FIG. 5. Error Prone PCR library construction procedure to randomize theFc region of trastuzumab-Fc5-2a.

FIG. 6. Sequences of isolated aglycosylated trastuzumab Fc variantsexhibiting high binding affinity to FcγRIIa over FcγRIIb. Spheroplastswere incubated with 20 nM of FcγRIIa-GST-Alexa488 and 100 nM ofFcγRIIb-GST for detection. FACS mean values are indicated in theparenthesis.

FIG. 7. Histogram showing fluorescence signals of aglycosylatedtrastuzumab Fc1001 variants in comparison with wild type aglycosylatedtrastuzumab and aglycosylated trastuzumab Fc5-2a. Spheroplasts wereincubated with 20 nM of FcγRIIa-GST-Alexa488 and 100 nM of FcγRIIb-GSTfor FACS analysis. M: Mean fluorescence intensity.

FIG. 8. Mutation points of isolated aglycosylated trastuzumab-Fc1001represented on the 3D structure of glycosylated IgG (PBD Code: 1FC1).

FIG. 9. SDS-PAGE gels showing the purified aglycosylated Fc2a,aglycosylated Fc1001, aglycosylated Fc1003, aglycosylated Fc1002 andaglycosylated Fc1004 produced in HEK293 cells. Lane M: molecular weightstandard; lane 1: Fc1001; lane 2: Fc1002; lane 3: Fc1004; lane 4: Fc2a;lane 1a: Fc1003.

FIG. 10. SPR sensorgrams for aglycosylated trastuzumab format antibodies(AglycoT) of Fc variants to FcγRIIa and FcγRIIb. (A-H) SPR sensorgramsof (A) AglycoT-Fc2a for binding to FcγRIIa-GST, (B) AglycoT-Fc2a forbinding to FcγRIIb-GST, (C) AglycoT-Fc1001 for binding to FcγRIIa-GST,(D) AglycoT-Fc1001 for binding to FcγRIIb-GST, (E) AglycoT-Fc 1003 forbinding to FcγRIIa-GST, (F) AglycoT-Fc 1003 for binding to FcγRIIb-GST,(G) AglycoT-Fc1004 for binding to FcγRIIa-GST, and (H) AglycoT-Fc1004for binding to FcγRIIb-GST.

FIG. 11. SDS-PAGE showing the purified FcγRIIb-strep isolated fromHEK293F cells. Lane M: molecular weight standard; lane 1: FcγRIIb-strepno denaturing; lane 2: FcγRIIb-strep denatured at 100° C. for 5 minutes.

FIG. 12. ELISA assay showing higher affinity of tetrameric FcγRIIb-Strepfusion for human IgG than dimeric FcγRIIb-GST fusion.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The inventors previously overcame several major problems with currentimmunotherapeutic technologies in providing aglycosylated antibody Fcdomains that are able to bind to Fc receptor polypeptides. Additional Fcdomains with engineered properties have been developed. Furtherembodiments and advantages are described below, though information aboutFc libraries and screening methods are provided.

I. PERIPLASMIC EXPRESSION

In some embodiments, polypeptide comprising an antibody Fc domain may beexpressed in the periplasmic space of a gram negative bacteria.Furthermore, in some aspects an antibody Fc domain may be anchored tothe periplasmic face of the inner membrane. For example, an Fc domainmay be directly fused to a membrane spanning or membrane boundpolypeptide or may interact (e.g., via protein-protein interactions)with a membrane spanning or membrane bound polypeptide. Such a techniquemay be termed “Anchored Periplasmic Expression” or “APEx”.

The periplasmic compartment is contained between the inner and outermembranes of Gram negative cells (see, e.g., Oliver, 1996). As asub-cellular compartment, it is subject to variations in size, shape andcontent that accompany the growth and division of the cell. Within aframework of peptidoglycan heteroploymer is a dense mileau ofperiplasmic proteins and little water, lending a gel-like consistency tothe compartment (Hobot et al., 1984; van Wielink and Duine, 1990). Thepeptidoglycan is polymerized to different extents depending on theproximity to the outer membrane, close-up it forms the murein sacculusthat affords cell shape and resistance to osmotic lysis.

The outer membrane (see Nikaido, 1996) is composed of phospholipids,porin proteins and, extending into the medium, lipopolysaccharide (LPS).The molecular basis of outer membrane integrity resides with LPS abilityto bind divalent cations (Mg²⁺ and Ca²⁺) and link each otherelectrostatically to form a highly ordered quasi-crystalline ordered“tiled roof” on the surface (Labischinski et al., 1985). The membraneforms a very strict permeability barrier allowing passage of moleculesno greater than around 650 Da (Burman et al., 1972; Decad and Nikaido,1976) via the porins. The large water filled porin channels areprimarily responsible for allowing free passage of mono anddisaccharides, ions and amino acids in to the periplasm compartment(Nikaido and Nakae, 1979; Nikaido and Vaara, 1985). With such strictphysiological regulation of access by molecules to the periplasm it mayappear, at first glance, inconceivable that large ligands (i.e., largerthan the 650 Da exclusion limit) could be employed in screening methods.However, the inventors have shown that ligands greater than 2000 Da insize can diffuse into the periplasm without disruption of theperiplasmic membrane. Such diffusion can be aided by one or moretreatments of a bacterial cell, thereby rendering the outer membranemore permeable, as is described herein below.

Method for expressing polypeptides and in particular antibodies in theperiplasmic space are known in the art for example see U.S. Pat. No.7,094,571 and U.S. Patent Publ. 20030180937 and 20030219870 eachincorporated herein by reference. In some cases, a gram negativebacterial cell of the invention may be defined as an E. coli cell.Furthermore, in some aspects a Gram negative bacterial cell may bedefined as a genetically engineered bacterial cell such as a Jude-1strain of E. coli.

II. PERMEABILIZATION OF THE OUTER MEMBRANE

In some embodiments, methods involve disrupting, permeablizing orremoving the outer membrane of bacteria are well known in the art, forexample, see U.S. Pat. No. 7,094,571. For instance, prior to contactingthe bacterial cells with an FcR polypeptide the outer membrane of thebacterial cell may be treated with hyperosmotic conditions, physicalstress, lysozyme, EDTA, a digestive enzyme, a chemical that disrupts theouter membrane, or by infecting the bacterium with a phage or acombination of the foregoing methods. Thus, in some cases, the outermembrane may be disrupted by lysozyme and EDTA treatment. Furthermore,in certain embodiments, the bacterial outer membrane may be removedentirely.

In one embodiment, methods are employed for increasing the permeabilityof the outer membrane to one or more labeled ligands. This can allowscreening access of labeled ligands otherwise unable to cross the outermembrane. However, certain classes of molecules, for example,hydrophobic antibiotics larger than the 650 Da exclusion limit, candiffuse through the bacterial outer membrane itself, independent ofmembrane porins (Farmer et al., 1999). The process may actuallypermeabilize the membrane on so doing (Jouenne and Junter, 1990). Such amechanism has been adopted to selectively label the periplasmic loops ofa cytoplasmic membrane protein in vivo with a polymyxin B nonapeptide(Wada et al., 1999). Also, certain long chain phosphate polymers (100Pi) appear to bypass the normal molecular sieving activity of the outermembrane altogether (Rao and Torriani, 1988).

Conditions have been identified that lead to the permeation of ligandsinto the periplasm without loss of viability or release of the expressedproteins from the cells, but the invention may be carried out withoutmaintenance of the outer membrane. As demonstrated herein Fc domainsexpressed or anchored candidate binding polypeptides in the periplasmicspace the need for maintenance of the outer membrane (as a barrier toprevent the leakage of the biding protein from the cell) to detect boundlabeled ligand is removed. As a result, cells expressing bindingproteins anchored to the outer (periplasmic) face of the cytoplasmicmembrane can be fluorescently labeled simply by incubating with asolution of fluorescently labeled ligand in cells that either have apartially permeabilized membrane or a nearly completely removed outermembrane.

The permeability of the outer membrane of different strains of bacterialhosts can vary widely. It has been shown previously that increasedpermeability due to OmpF overexpression was caused by the absence of ahistone like protein resulting in a decrease in the amount of a negativeregulatory mRNA for OmpF translation (Painbeni et al., 1997). Also, DNAreplication and chromosomal segregation is known to rely on intimatecontact of the replisome with the inner membrane, which itself contactsthe outer membrane at numerous points. A preferred host for libraryscreening applications is E. coli ABLEC strain, which additionally hasmutations that reduce plasmid copy number.

Treatments such as hyperosmotic shock can improve labelingsignificantly. It is known that many agents including, calcium ions(Bukau et al., 1985) and even Tris buffer (Irvin et al., 1981) alter thepermeability of the outer-membrane. Further, phage infection stimulatesthe labeling process. Both the filamentous phage inner membrane proteinpIII and the large multimeric outer membrane protein pIV can altermembrane permeability (Boeke et al., 1982) with mutants in pIV known toimprove access to maltodextrins normally excluded (Marciano et al.,1999). Using the techniques of the invention, comprising a judiciouscombination of strain, salt and phage, a high degree of permeability maybe achieved (Daugherty et al., 1999). Cells comprising anchored orperiplasm-associated polypeptides bound to fluorescently labeled ligandscan then be easily isolated from cells that express binding proteinswithout affinity for the labeled ligand using flow cytometry or otherrelated techniques. However, in some cases, it will be desired to useless disruptive techniques in order to maintain the viability of cells.EDTA and Lysozyme treatments may also be useful in this regard.

III. ANTIBODY-BINDING POLYPEPTIDES

In certain aspects there are methods for identifying antibody Fc domainswith a specific affinity for antibody-binding polypeptide such as an Fcreceptor. In some embodiments, an Fc domain is engineered to bind one ormore specific Fc receptors. Additionally or alternatively, an Fc domainmay be engineered so that it does not specifically bind one or morespecific Fc receptors.

In certain embodiments, there are compositions comprising aproteinaceous molecule that has been modified relative to a native orwild-type protein.

In some embodiments that proteinaceous compound has been deleted ofamino acid residues; in other embodiments, amino acid residues of theproteinaceous compound have been replaced, while in still furtherembodiments both deletions and replacements of amino acid residues inthe proteinaceous compound have been made. Furthermore, a proteinaceouscompound may include an amino acid molecule comprising more than onepolypeptide entity. As used herein, a “proteinaceous molecule,”“proteinaceous composition,” “proteinaceous compound,” “proteinaceouschain” or “proteinaceous material” generally refers, but is not limitedto, a protein of greater than about 200 amino acids or the full lengthendogenous sequence translated from a gene; a polypeptide of 100 aminoacids or greater; and/or a peptide of 3 to 100 amino acids. All the“proteinaceous” terms described above may be used interchangeablyherein; however, it is specifically contemplated that embodiments may belimited to a particular type of proteinaceous compound, such as apolypeptide. Furthermore, these terms may be applied to fusion proteinsor protein conjugates as well. A protein may include more than onepolypeptide. An IgG antibody, for example, has two heavy chainpolypeptides and two light chain polypeptides, which are joined to eachother through disulfide bonds.

As used herein a “distinct Fc domain” may be defined as a domain thatdiffers from another Fc by as little as one amino acid. Methods formaking a library of distinct antibody Fc domains or nucleic acids thatencode antibodies are well known in the art and exemplified herein. Forexample, in some cases Fc domains may be amplified by error prone PCR asexemplified herein. Furthermore, in certain cases a plurality ofantibody Fc domains may comprise a stretch (1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more) amino acids that have been randomized. In certain casesspecific mutations may be engineered into Fc domains. For example, insome aspects, residues that are normally glycosylated in an antibody Fcdomain may be mutated. Furthermore, in certain aspects, residues thatare normally glycosylated (or adjacent residues) may be used as a sitefor an insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.An amino acid insertion may be made at, or adjacent to, a residuecorresponding to amino acid 384 of the IgG1 Fc (SEQ ID NO:2). In stillfurther cases, a population of gram negative bacteria according to theinvention may be defined as comprising at least about 1×10³, 1×10⁴,1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, or more distinct antibodies Fc domains. Insome specific cases, a population of Gram negative bacterial cells maybe produced by a method comprising the steps of: (a) preparing aplurality of nucleic acid sequences encoding a plurality of distinctantibody Fc domains; and (b) transforming a population of Gram negativebacteria with said nucleic acids wherein the Gram negative bacteriacomprise a plurality of antibody Fc domains expressed in the periplasm.

A variety of antibody-binding domains (e.g., FcR polypeptides) are knownin the art and may be used in the methods and compositions of theinvention. For example, in some aspects, an FcR may have specificity fora particular type or subtype of Ig, such as IgA, IgM, IgE or IgG (e.g.,IgG1, IgG2a, IgG2b, IgG3 or IgG4). Thus, in some embodiments theantibody-binding domain may be defined as an IgG binding domain. The FcRpolypeptide may compries an eukaryotic, prokaryotic, or synthetic FcRdomain. For instance, an antibody Fc-binding domain may be defined as amammalian, bacterial or synthetic binding domain. Some Fc-bindingdomains for use in the invention include but are not limited to abinding domain from one of the polypeptides of Table 1. For example, anFc-binding polypeptide may be encoded by an FCGR2A, FCGR2B, FCGR2c,FCGR3A, FCGR3B, FCGR1A, Fcgr1, FCGR2, FCGR2, Fcgr2, Fcgr2, FCGR3, FCGR3,Fcgr3, FCGR3, Fcgr3, FCGRT, mrp4, spa or spg gene. Preferably, an FcRpolypeptide for use according to the invention may be an Fc bindingregion from human FcγRIa, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb,FcαRI or C1q.

In still further embodiments of the invention an Fc polypeptide may beanchored to the inner membrane of a Gram negative bacteria. Methods andcompositions for the anchoring of polypeptides to the inner membrane ofGram negative bacterial have previously been described (U.S. Pat. No.7,094,571 and U.S. Patent Publ. 20050260736). Thus, in some aspects, anFc domain may be fused to a polypeptide that is associated with orintegrated in a bacterial inner membrane. Such a fusion protein maycomprise an N terminal or C terminal fusion with an Fc domain and insome case may comprise additional linker amino acids between themembrane anchoring polypeptide and the Fc domain. In certain specificcases, a membrane anchoring polypeptide may be the first six amino acidsencoded by the E. coli NlpA gene, one or more transmembrane α-helicesfrom an E. coli inner membrane protein, a gene III protein offilamentous phage or a fragment thereof, or an inner membranelipoprotein or fragment thereof. Thus, as an example, a membraneanchoring polypeptide may be an inner membrane lipoprotein or fragmentthereof such as from AraH, MglC, MalF, MalG, MalC, MalD, RbsC, RbsC,ArtM, ArtQ, GlnP, ProW, HisM, HisQ, LivH, LivM, LivA, LivE, DppB, DppC,OppB, AmiC, AmiD, BtuC, ThuD, FecC, FecD, FecR, FepD, NikB, NikC, CysT,CysW, UgpA, UgpE, PstA, PstC, PotB, PotC, PotH, Pod, ModB, NosY, PhnM,LacY, SecY, TolC, Dsb, B, DsbD, TouB, TatC, CheY, TraB, ExbD, ExbB orAas.

The skilled artisan will understand that methods for selecting cellsbased upon their interaction (binding) with an FcR are well known in theart. For example, an FcR may be immobilized on a column or bead (e.g., amagnetic bead) and the bacterial cell binding to the FcR separated byrepeated washing of the bead (e.g., magnetic separation) or column.Furthermore, in some aspects a target ligand may be labeled such as witha fluorophor, a radioisotope or an enzyme. Thus, bacterial cells may, insome cases, be selected by detecting a label on a bound FcR. Forexample, a fluorophore may be used to select cells using fluorescenceactivated cell sorting (FACS). Furthermore, in some aspects, bacterialcells may be selected based on binding or lack of binding two or moreFcR polypeptides. For instance, bacteria may be selected that displayantibodies that bind to two FcR polypeptides, wherein each FcR is usedto select the bacterial sequentially. Conversely, in certain aspects,bacteria may be selected that display antibody Fc domains that bind toone FcR (such as an FcR comprising a first label) but not to a secondFcR (e.g., comprising a second label). The foregoing method may be used,for example, to identify antibody Fc domains that bind to a specific FcRbut not a second specific FcR.

In certain embodiments the size of the at least one Fc polypeptideproteinaceous molecule may comprise, but is not limited to, about or atleast 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000 or greater amino molecule residues, and any rangederivable therein. Compounds may include the above-mentioned number ofcontiguous amino acids from SEQ ID NO:1 (human IgG Fc polypeptide) orfrom SEQ ID NOs 4-9 and these may be further qualified as having apercent identity or homology to SEQ ID NO:2 or any of SEQ ID NO:4-9(discussed below). It is contemplated that embodiments with respect toSEQ ID NO:2 may be employed with respect to any other amino acidsequences described herein, and vice versa, if appropriate.

As used herein, an “amino molecule” refers to any amino acid, amino acidderivative or amino acid mimic as would be known to one of ordinaryskill in the art. In certain embodiments, the residues of theproteinaceous molecule are sequential, without any non-amino moleculeinterrupting the sequence of amino molecule residues. In otherembodiments, the sequence may comprise one or more non-amino moleculemoieties. In particular embodiments, the sequence of residues of theproteinaceous molecule may be interrupted by one or more non-aminomolecule moieties.

A. Modified Proteins and Polypeptides

Embodiments concerns modified proteins and polypeptides, particularly amodified protein or polypeptide that exhibits at least one functionalactivity that is comparable to the unmodified version, yet the modifiedprotein or polypeptide possesses an additional advantage over theunmodified version, such as provoking ADCC, easier or cheaper toproduce, eliciting fewer side effects, and/or having better or longerefficacy or bioavailability. Thus, when the present application refersto the function or activity of “modified protein” or a “modifiedpolypeptide” one of ordinary skill in the art would understand that thisincludes, for example, a protein or polypeptide that 1) performs atleast one of the same activities or has at least one of the samespecificities as the unmodified protein or polypeptide, but that mayhave a different level of another activity or specificity; and 2)possesses an additional advantage over the unmodified protein orpolypeptide. Determination of activity may be achieved using assaysfamiliar to those of skill in the art, particularly with respect to theprotein's activity, and may include for comparison purposes, forexample, the use of native and/or recombinant versions of either themodified or unmodified protein or polypeptide. It is specificallycontemplated that embodiments concerning a “modified protein” may beimplemented with respect to a “modified polypeptide,” and vice versa. Inaddition to the modified proteins and polypeptides discussed herein,embodiments may involve domains, polypeptides, and proteins described inWO 2008/137475, which is hereby specifically incorporated by reference.

Modified proteins may possess deletions and/or substitutions of aminoacids; thus, a protein with a deletion, a protein with a substitution,and a protein with a deletion and a substitution are modified proteins.In some embodiments these modified proteins may further includeinsertions or added amino acids, such as with fusion proteins orproteins with linkers, for example. A “modified deleted protein” lacksone or more residues of the native protein, but possesses thespecificity and/or activity of the native protein. A “modified deletedprotein” may also have reduced immunogenicity or antigenicity. Anexample of a modified deleted protein is one that has an amino acidresidue deleted from at least one antigenic region—that is, a region ofthe protein determined to be antigenic in a particular organism, such asthe type of organism that may be administered the modified protein.

Substitutional or replacement variants typically contain the exchange ofone amino acid for another at one or more sites within the protein andmay be designed to modulate one or more properties of the polypeptide,particularly its effector functions and/or bioavailability.Substitutions may or may not be conservative, that is, one amino acid isreplaced with one of similar shape and charge. Conservativesubstitutions are well known in the art and include, for example, thechanges of: alanine to serine; arginine to lysine; asparagine toglutamine or histidine; aspartate to glutamate; cysteine to serine;glutamine to asparagine; glutamate to aspartate; glycine to proline;histidine to asparagine or glutamine; isoleucine to leucine or valine;leucine to valine or isoleucine; lysine to arginine; methionine toleucine or isoleucine; phenylalanine to tyrosine, leucine or methionine;serine to threonine; threonine to serine; tryptophan to tyrosine;tyrosine to tryptophan or phenylalanine; and valine to isoleucine orleucine.

In addition to a deletion or substitution, a modified protein maypossess an insertion of residues, which typically involves the additionof at least one residue in the polypeptide. This may include theinsertion of a targeting peptide or polypeptide or simply a singleresidue. Terminal additions, called fusion proteins, are discussedbelow.

The term “biologically functional equivalent” is well understood in theart and is further defined in detail herein. Accordingly, sequences thathave between about 70% and about 80%, or between about 81% and about90%, or even between about 91% and about 99% of amino acids that areidentical or functionally equivalent to the amino acids of a nativepolypeptide are included, provided the biological activity of theprotein is maintained. A modified protein may be biologicallyfunctionally equivalent to its native counterpart.

It also will be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

The following is a discussion based upon changing of the amino acids ofa protein to create an equivalent, or even an improved,second-generation molecule. For example, certain amino acids may besubstituted for other amino acids in a protein structure with or withoutappreciable loss of interactive binding capacity with structures suchas, for example, binding sites to substrate molecules. Since it is theinteractive capacity and nature of a protein that defines that protein'sbiological functional activity, certain amino acid substitutions can bemade in a protein sequence, and in its underlying DNA coding sequence,and nevertheless produce a protein with like properties. It is thuscontemplated by the inventors that various changes may be made in theDNA sequences of genes without appreciable loss of their biologicalutility or activity, as discussed below. A proteinaceous molecule has“homology” or is considered “homologous” to a second proteinaceousmolecule if one of the following “homology criteria” is met: 1) at least30% of the proteinaceous molecule has sequence identity at the samepositions with the second proteinaceous molecule; 2) there is somesequence identity at the same positions with the second proteinaceousmolecule and at the nonidentical residues, at least 30% of them areconservative differences, as described herein, with respect to thesecond proteinaceous molecule; or 3) at least 30% of the proteinaceousmolecule has sequence identity with the second proteinaceous molecule,but with possible gaps of nonidentical residues between identicalresidues. As used herein, the term “homologous” may equally apply to aregion of a proteinaceous molecule, instead of the entire molecule. Ifthe term “homology” or “homologous” is qualified by a number, forexample, “50% homology” or “50% homologous,” then the homology criteria,with respect to 1), 2), and 3), is adjusted from “at least 30%” to “atleast 50%.” Thus it is contemplated that there may homology of at least30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, ormore between two proteinaceous molecules or portions of proteinaceousmolecules.

Alternatively, a modified polypeptide may be characterized as having acertain percentage of identity to an unmodified polypeptide or to anypolypeptide sequence disclosed herein, including SEQ ID NO:1 or any ofSEQ ID NOs:4-9. The percentage identity may be at most or at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%(or any range derivable therein) between two proteinaceous molecules orportions of proteinaceous molecules. It is contemplated that percentageof identity discussed above may relate to a particular region of apolypeptide compared to an unmodified region of a polypeptide. Forinstance, a polypeptide may contain a modified or mutant Fc domain thatcan be characterized based on the identity of the amino acid sequence ofthe modified or mutant Fc domain to an unmodified or mutant Fc domainfrom the same species. A modified or mutant human Fc domaincharacterized, for example, as having 90% identity to an unmodified Fcdomain means that 90% of the amino acids in that domain are identical tothe amino acids in the unmodified human Fc domain (SEQ ID NO:1).

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte & Doolittle, 1982). It is accepted that therelative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine(−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still produce a biologicallyequivalent and immunologically equivalent protein. In such changes, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions generally are based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take into consideration the variousforegoing characteristics are well known to those of skill in the artand include: arginine and lysine; glutamate and aspartate; serine andthreonine; glutamine and asparagine; and valine, leucine and isoleucine.

A variety of Fc receptors to which Fc domains bind are well known in theart and some examples of receptors are listed below in Table 1.

TABLE 1 Selected FcR Polypeptides Protein name Gene name DescriptionOrganisms Length (aa) Reference Fc-gamma FCGR2A Low affinity Homosapiens 317 (Stuart et al., RII-a immunoglobulin (Human) 1987) (CD32)gamma Fc region receptor II-a precursor Fc-gamma FCGR2A Low affinity Pan316 RII-a immunoglobulin troglodytes gamma Fc (Chimpanzee) regionreceptor II-a precursor Fc-gamma FCGR2B Low affinity Homo sapiens 310(Stuart et al., RII-b immunoglobulin (Human) 1989) gamma Fc regionreceptor II-b precursor Fc-gamma FCGR2C Low affinity Homo sapiens 323(Stuart et al., RII-c immunoglobulin (Human) 1989) gamma Fc regionreceptor II-c precursor Fc-gamma FCGR3A Low affinity Homo sapiens 254(Ravetch and RIIIa immunoglobulin (Human) Perussia, gamma Fc 1989)region receptor III-A precursor Fc-gamma FCGR3B Low affinity Homosapiens 233 (Ravetch and RIIIb immunoglobulin (Human) Perussia, gamma Fc1989) region receptor III-B precursor Fc-gamma FCGR1A High affinity Homosapiens 374 (Allen and RI (CD64) immunoglobulin (Human) Seed, 1988)gamma Fc receptor I precursor Fc-gamma Fcgr1 High affinity Mus musculus404 (Sears et al., RI immunoglobulin (Mouse) 1990) gamma Fc receptor Iprecursor Fc-gamma FCGR2 Low affinity Bos taurus 296 (Zhang et al., RIIimmunoglobulin (Bovine) 1994) gamma Fc region receptor II precursorFc-gamma FCGR2 Low affinity Cavia 341 (Tominaga et RII immunoglobulinporcellus al., 1990) gamma Fc (Guinea pig) region receptor II precursorFc-gamma Fcgr2 Low affinity Mus musculus 330 (Ravetch et RIIimmunoglobulin (Mouse) al., 1986) gamma Fc region receptor II precursorFc-gamma Fcgr2 Low affinity Rattus 285 (Bocek and RII immunoglobulinnorvegicus Pecht, 1993) gamma Fc (Rat) region receptor II precursorFc-gamma FCGR3 Low affinity Bos taurus 250 (Collins et RIIIimmunoglobulin (Bovine) al., 1997) gamma Fc region receptor IIIprecursor Fc-gamma FCGR3 Low affinity Macaca 254 RIII immunoglobulinfascicularis gamma Fc (Crab eating region receptor macaque) IIIprecursor (Cynomolgus monkey) Fc-gamma Fcgr3 Low affinity Mus musculus261 (Ravetch et RIII immunoglobulin (Mouse) al., 1986) gamma Fc regionreceptor III precursor Fc-gamma FCGR3 Low affinity Sus scrofa 257(Halloran et RIII immunoglobulin (Pig) al., 1994) gamma Fc regionreceptor III precursor Fc-gamma Fcgr3 Low affinity Rattus 267 (Zeger etal., RIII immunoglobulin norvegicus 1990) gamma Fc (Rat) region receptorIII precursor FcRn FCGRT IgG receptor Homo sapiens 365 transporter(Human) FcRn large subunit p51 precursor FcRn FCGRT IgG receptor Macaca365 transporter fascicularis FcRn large (Crab eating subunit p51macaque) precursor (Cynomolgus monkey) FcRn Fcgrt IgG receptor Musmusculus 365 (Ahouse et transporter (Mouse) al., 1993) FcRn largesubunit p51 precursor FcRn Fcgrt IgG receptor Rattus 366 (Simister andtransporter norvegicus Mostov, FcRn large (Rat) 1989) subunit p51precursor MRP mrp4 Fibrinogen- Streptococcus 388 (Stenberg et proteinand Ig-binding pyogenes al., 1992) protein precursor Protein B cAMPfactor Streptococcus 226 (Ruhlmann et agalactiae al., 1988) protein Aspa Immunoglobulin Staphylococcus 516 (Uhlen et al., G-binding aureus(strain 1984) protein A NCTC 8325) precursor protein A spaImmunoglobulin Staphylococcus 508 (Shuttleworth G-binding aureus et al.,1987) protein A precursor protein A spa Immunoglobulin Staphylococcus450 (Kuroda et G-binding aureus (strain al., 2001) protein A Mu50/ATCCprecursor 700699) protein A spa Immunoglobulin Staphylococcus 450(Kuroda et G-binding aureus (strain al., 2001) protein A N315) precursorprotein G spg Immunoglobulin Streptococcus 448 (Fahnestock G-binding sp.group G et al., 1986) protein G precursor protein G spg ImmunoglobulinStreptococcus 593 (Olsson et al., G-binding sp. group G 1987) protein Gprecursor protein H Immunoglobulin Streptococcus 376 (Gomi et al.,G-binding pyogenes 1990) protein H serotype M1 precursor Protein sbi sbiImmunoglobulin Staphylococcus 436 (Zhang et al., G-binding aureus(strain 1998) protein sbi NCTC 8325-4) precursor Allergen Allergen Aspfl Aspergillus  32 Asp fl 1 1 causes an flavus allergic reaction inhuman. Binds to IgE and IgG Allergen Allergen Asp fl Aspergillus  20 Aspfl 2 2 causes an flavus allergic reaction in human. Binds to IgE and IgGAllergen Allergen Asp fl Aspergillus  32 Asp fl 3 3 causes an flavusallergic reaction in human. Binds to IgE and IgG Fc-epsilon IgE receptorHomo sapiens RI displayed on (Human) Mast cells, Eosinophils andBasophils Fc-alpha RI IgA (IgA1, Homo sapiens (CD86) IgA2) receptor(Human) displayed on Macrophages C1q C1QA C1q is Homo sapiensNP_057075.1, multimeric (Human) C1QB complex that NP_000482.3, binds toC1QC antibody Fc NP_758957.1  composed of 6 A chains, 6 B chains and 6 Cchains

As discussed above, a polypeptide may comprise an aglycosylated antibodyFc domain capable of binding an FcR polypeptide. In some aspects, theaglycosylated Fc domain may be further defined as having a specificaffinity for an FcR polypeptide under physiological conditions. Forinstance an Fc domain may have an equilibrium dissociation constantbetween about 10⁻⁶ M to about 10⁻⁹ M under physiological conditions.Furthermore in some aspects an aglycosylated Fc domain may be defined ascomprising one or more amino acid substitution or insertion relative toa wild-type sequence, such as a human wild-type sequence.

Means of preparing such a polypeptide include those discussed in WO2008/137475, which is hereby incorporated by reference. One canalternatively prepare such polypeptides directly by genetic engineeringtechniques such as, for example, by introducing selected amino acidsubstitutions or insertions into a known Fc background, wherein theinsertion or substitution provides an improved FcR binding capability toaglycosylated Fc regions, as discussed above.

In some embodiments, an aglycosylated Fc domain comprises a specificbinding affinity for an FcR such as human FcγRIa, FcγRIIa, FcγRIIb,FcγRIIc, FcγRIIIa, FcγRIIIb, FcαRI or C1q. Thus, in some aspects anaglycosylated Fc domain of the invention is defined as an Fc domain witha specific affinity for FcγRIa. Furthermore, such an Fc domain may bedefined as having an equilibrium dissociation constant, with respect toFcγRIa binding, of about 10⁻⁶ M to about 10⁻⁹ M under physiologicalconditions.

B. Modified Antibodies and Proteinaceous Compounds with HeterologousRegions

Embodiments concern an Fc polypeptide proteinaceous compound that mayinclude amino acid sequences from more than one naturally occurring ornative polypeptides or proteins. Embodiments discussed above arecontemplated to apply to this section, and vice versa. For instance, amodified antibody is one that contains a modified Fc domain with anantigen binding domain. Moreover, the antibody may have two differentantigen binding regions, such as a different region on each of the twoheavy chains. Alternatively or additionally, in some embodiments, thereare polypeptides comprising multiple heterologous peptides and/orpolypeptides (“heterologous” meaning they are not derived from the samepolypeptide). A proteinaceous compound or molecule, for example, couldinclude a modified Fc domain with a protein binding region that is notfrom an antibody. In some embodiments, there are polypeptides comprisinga modified Fc domain with a protein binding region that binds acell-surface receptor. These proteinaceous molecule comprising multiplefunctional domains may be two or more domains chemically conjugated toone another or it may be a fusion protein of two or more polypeptidesencoded by the same nucleic acid molecule. It is contemplated thatproteins or polypeptides may include all or part of two or moreheterologous polypeptides.

Thus, a multipolypeptide proteinaceous compound may be comprised of allor part of a first polypeptide and all or part of a second polypeptide,a third polypeptide, a fourth polypeptide, a fifth polypeptide, a sixthpolypeptide, a seventh polypeptide, an eight polypeptide, a ninthpolypeptide, a tenth polypeptide, or more polypeptides.

Polypeptides or proteins (including antibodies) having an antigenbinding domain or region of an antibody and an aglycosylated Fc domaincan be used against any antigen or epitope, including but not limited toproteins, subunits, domains, motifs, and/or epitopes belonging to thefollowing list of targets: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a,8-oxo-dG, A1 Adenosine Receptor, A33, ACE, ACE-2, Activin, Activin A,Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2,Activin RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12,ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Addressins,aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V/beta-1antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART,Artemin, anti-Id, ASPARTIC, Atrial natriuretic factor, av/b3 integrin,Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte Stimulator (BlyS), BACE,BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA,BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a,BMP-3 Osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8(BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1,BMPR-II (BRK-3), BMPs, b-NGF, BOK, Bombesin, Bone-derived neurotrophicfactor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a,C10, CA125, CAD-8, Calcitonin, cAMP, carcinoembryonic antigen (CEA),carcinoma-associated antigen, Cathepsin A, Cathepsin B, CathepsinC/DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O,Cathepsin S, Cathepsin V, Cathepsin X/ZIP, CBL, CCI, CCK2, CCL, CCL1,CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2,CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3,CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2,CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5,CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18,CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L,CD32, CD33 (p67 proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46,CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1),CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152,CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin,Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1,COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL,CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10,CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2,CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN,DCC, DcR3, DC-SIGN, Decay accelerating factor, des(1-3)-IGF-I (brainIGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA,EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelinreceptor, Enkephalinase, eNOS, Eot, eotaxin1, EpCAM, Ephrin B2/EphB4,EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc,Factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1,Ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, Fibrin, FL,FLIP, Flt-3, Flt-4, Follicle stimulating hormone, Fractalkine, FZD1,FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6,GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14,CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8(Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1,GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut 4, glycoproteinIIb/IIIa (GP IIb/IIIa), GM-CSF, gp130, gp72, GRO, Growth hormonereleasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gBenvelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL,Hemopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu(ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gBglycoprotein, HSV gD glycoprotein, HGFA, High molecular weightmelanoma-associated antigen (HMW-MM), HIV gp120, HIV IIIB gp120 V3 loop,HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, humancytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP,ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGFbinding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2,IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12,IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta,INF-gamma, Inhibin, iNOS, Insulin A-chain, Insulin B-chain, Insulin-likegrowth factor 1, integrin alpha2, integrin alpha3, integrin alpha4,integrin alpha4/beta1, integrin alpha4/beta7, integrin alpha5 (alphaV),integrin alpha5/beta1, integrin alpha5/beta3, integrin alpha6, integrinbeta1, integrin beta2, interferon gamma, IP-10, I-TAC, JE, Kallikrein 2,Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12, Kallikrein 14,Kallikrein 15, Kallikrein L1, Kallikrein L2, Kallikrein L3, KallikreinL4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin 5, LAMP, LAP, LAP(TGF-1), Latent TGF-1, Latent TGF-1 bp1, LBP, LDGF, LECT2, Lefty,Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT,lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b, LTB4, LTBP-1, Lungsurfactant, Luteinizing hormone, Lymphotoxin Beta Receptor, Mac-1,MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer,METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP,MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13,MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo,MSK, MSP, mucin (Muc1), MUC18, Muellerian-inhibitin substance, Mug,MuSK, NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin,Neurotrophin-3, -4, or -6, Neurturin, Neuronal growth factor (NGF),NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN,OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid hormone, PARC, PARP,PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4,PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP),P1GF, PLP, PP14, Proinsulin, Prorelaxin, Protein C, PS, PSA, PSCA,prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51,RANK, RANKL, RANTES, RANTES, Relaxin A-chain, Relaxin B-chain, renin,respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factors,RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, Serum albumin, sFRP-3,Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat,STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72),TARC, TCA-3, T-cell receptors (e.g., T-cell receptor alpha/beta), TdT,TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkalinephosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific,TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII,TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, Thrombin, ThymusCk-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, Tissue Factor,TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc,TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID),TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI),TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROYTAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60),TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNFRIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50),TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6),TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 Ligand,TL2), TNFSF11 (TRANCE/RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3Ligand, DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1,THANK, TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15 (TL1A/VEGI),TNFSF18 (GITR Ligand AITR Ligand, TL6), TNFSF1A (TNF-a Conectin, DIF,TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4(OX40 Ligand gp34, TXGP1), TNFSF5 (CD40 Ligand CD154, gp39, HIGM1, IMD3,TRAP), TNFSF6 (Fas Ligand Apo-1 Ligand, APT1 Ligand), TNFSF7 (CD27Ligand CD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1BB Ligand CD137Ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE,transferring receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associatedantigen CA 125, tumor-associated antigen expressing Lewis Y relatedcarbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1,VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3(fit-4), VEGI, VIM, Viral antigens, VLA, VLA-1, VLA-4, VNR integrin, vonWillebrands factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4,WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B,WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD,and receptors for hormones and growth factors. In some embodiments, apolypeptide or protein has an antigen binding domain specific for one ormore cell surface tumor antigens. Methods and compositions may beemployed to target a tumor cell for ADCC.

Fc domains can bind to an FcR, however, it is contemplated that ADCC canbe directed not only through an antigen binding domain on thepolypeptide containing the Fc domain, but through some other proteinbinding domain. Consequently, embodiments concern an Fc domain and aheterologous non-antigen binding domain. In certain embodiments, thenon-antigen binding domain bind to the cell surface. Therefore, theseagents require either chemical conjugation to or fusion withagents/proteins which are capable of binding to specific target cells.Embodiments further include adjoining all or part of an aglycosylated Fcdomain to all or part of any of the proteins listed in Table 2. It iscontemplated that embodiments include, but are not limited to, theexamples provided in Table 2 and the description herein.

TABLE 2 Protein Genus Subgenus Species Subspecies 1) AntibodiesPolyclonal Monoclonal non-recombinant Recombinant chimeric single chaindiabody multimeric 2) Ligands for IL-1, IL-2, IL-3, cell-surface IL-4,IL-5, IL-6, receptors IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, Cytokines/ IL-19 growth factorsCytokines/growth factors for receptor tyrosine kinases GM-CSF, G-CSF,M-CSF, EGF, VEGF, FGF, PDGF, HGF, GDNF, Trk, AXL, LTK, TIE, ROR, DDR,KLG, RYK, MuSK ligands 3) Non-Ab binding protein for cell-surfacemolecule Binders of cell surface proteins Cluster of differentiation(CD) molecules

A ligand for receptor may be employed to target a cell expressing on itssurface the receptor for the ligand. Ligands also include, for instance,CD95 ligand, TRAIL, TNF (such as TNF-α. or TNF-β), growth factors,including those discussed above, such as VEGF and cytokines, such asinterferons or interleukins and variants thereof.

Embodiments with multiple domains are also contemplated, such as a VEGFTrap fusion protein that includes the second extracellular domain of theVEGF receptor 1 (Flt-1) with the third domain of the VEGF receptor 2(KDR/FIK-1) and an IgG Fc region.

a. Fusion and Conjugated Proteins

A specialized kind of insertional variant is the fusion protein. Thismolecule generally has all or a substantial portion of the nativemolecule, linked at the N- or C-terminus, to all or a portion of asecond polypeptide.

Embodiments also concern conjugated polypeptides, such as translatedproteins, polypeptides and peptides, that are linked to at least oneagent to form a modified protein or polypeptide. In order to increasethe efficacy of molecules as diagnostic or therapeutic agents, it isconventional to link or covalently bind or complex at least one desiredmolecule or moiety. Such a molecule or moiety may be, but is not limitedto, at least one effector or reporter molecule. Effector moleculescomprise molecules having a desired activity, e.g., cytotoxic activity.Non-limiting examples of effector molecules which have been attached toantibodies include toxins, anti-tumor agents, therapeutic enzymes,radio-labeled nucleotides, antiviral agents, chelating agents,cytokines, growth factors, and oligo- or poly-nucleotides. By contrast,a reporter molecule is defined as any moiety that may be detected usingan assay. Non-limiting examples of reporter molecules which have beenconjugated to antibodies include enzymes, radiolabels, haptens,fluorescent labels, phosphorescent molecules, chemiluminescentmolecules, chromophores, luminescent molecules, photoaffinity molecules,colored particles or ligands, such as biotin.

Any antibody of sufficient selectivity, specificity or affinity may beemployed as the basis for an antibody conjugate. Such properties may beevaluated using conventional immunological screening methodology knownto those of skill in the art. Sites for binding to biological activemolecules in the antibody molecule, in addition to the canonical antigenbinding sites, include sites that reside in the variable domain that canbind pathogens, B-cell superantigens, the T cell co-receptor CD4 and theHIV-1 envelope (Sasso et al., 1989; Shorki et al., 1991; Silvermann etal., 1995; Cleary et al., 1994; Lenert et al., 1990; Berberian et al.,1993; Kreier et al., 1991). In addition, the variable domain is involvedin antibody self-binding (Kang et al., 1988), and contains epitopes(idiotopes) recognized by anti-antibodies (Kohler et al., 1989).

Certain examples of antibody conjugates are those conjugates in whichthe antibody is linked to a detectable label. “Detectable labels” arecompounds and/or elements that can be detected due to their specificfunctional properties, and/or chemical characteristics, the use of whichallows the antibody to which they are attached to be detected, and/orfurther quantified if desired. Another such example is the formation ofa conjugate comprising an antibody linked to a cytotoxic oranti-cellular agent, and may be termed “immunotoxins.”

Amino acids such as selectively-cleavable linkers, synthetic linkers, orother amino acid sequences may be used to separate proteinaceousmoieties.

IV. ANTIBODY FC LIBRARIES

Examples of techniques that could be employed in conjunction withembodiments for creation of diverse antibody Fc domains and/orantibodies comprising such domains may employ techniques similar tothose for expression of immunoglobulin heavy chain libraries describedin U.S. Pat. No. 5,824,520. Previously employed Fc libraries arediscussed in WO 2008/137475, which is specifically incorporated byreference.

V. SCREENING ANTIBODY FC DOMAINS

There are embodiments involving methods for identifying moleculescapable of binding to a particular FcR. They are described herein, aswell as in PCT Application WO 2008/137475, which is hereby specificallyincorporated by reference in its entirety. The binding polypeptidesscreened may comprise a large library of diverse candidate Fc domains,or, alternatively, may comprise particular classes of Fc domains (e.g.,engineered point mutations or amino acid insertions) selected with aneye towards structural attributes that are believed to make them morelikely to bind the target ligand. In one embodiment, the candidatebinding protein is an intact antibody, or a fragment or portion thereofcomprising an Fc domain.

To identify a candidate Fc domain capable of binding a target ligand,one may carry out the steps of: providing a population of Gram negativebacterial cells that express a distinct antibody Fc domain; admixing thebacteria or phages and at least a first labeled or immobilized targetligand (FcR polypeptide) capable of contacting the antibody andidentifying at least a first bacterium expressing a molecule capable ofbinding the target ligand.

In some aspects of the aforementioned method, the binding betweenantibody Fc domain and a labeled FcR polypeptide will prevent diffusingout of a bacterial cell. In this way, molecules of the labeled ligandcan be retained in the periplasm of the bacterium comprising apermeablized outer membrane. Alternatively, the periplasm can beremoved, whereby the Fc domain will cause retention of the boundcandidate molecule since Fc domains are shown to associate with theinner membrane. The labeling may then be used to isolate the cellexpressing a binding polypeptide capable of binding the FcR polypeptide,and in this way, the gene encoding the Fc domain polypeptide isolated.The molecule capable of binding the target ligand may then be producedin large quantities using in vivo or ex vivo expression methods, andthen used for any desired application, for example, for diagnostic ortherapeutic applications. Furthermore, it will be understood thatisolated antibody Fc domains identified may be used to construct anantibody fragment or full-length antibody comprising an antigen bindingdomain.

In further embodiments, methods for producing bacteria of the invention,may comprise at least two rounds of selection (step c) wherein thesub-population of bacterial cells obtained in the first round ofselection is subjected to at least a second round of selection based onthe binding of the candidate antibody Fc domain to an FcR. Furthermorein some aspects the sub-population of bacterial cells obtained in thefirst round of selection may be grown under permissive conditions priorto a second selection (to expand the total number of cells). Thus, insome aspects, methods may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or morerounds of selection. Furthermore, in some aspects, a sub-population ofbacterial cells obtained from each round of selection will be grownunder permissive conditions before a subsequent round of selection.Cells isolated following one or more such rounds of selection may besubjected to additional rounds of mutagenesis. In some cases, selectionwill be performed after removing FcR polypeptide that is not bound tothe antibody. Furthermore, in some cases the stringency of selection maybe modified by adjusting the pH, salt concentration, or temperature of asolution comprising bacteria that display antibodies. Thus, in someaspects, it may be preferred that a bacterial cell of the invention isgrown at a sub-physiological temperature such as at about 25° C.

In still further aspects, a method of producing a bacterial cellaccording to the invention may be further defined as a method ofproducing a nucleic acid sequence encoding an Fc domain that binds to atleast a first FcR. Thus, a bacterial cell produced by the methods hereinmay be used to clone a nucleic acid sequence encoding the Fc domainhaving a specific affinity for an FcR polypeptide. Methods for isolatingand amplifying such a nucleic acid from a cell for example by PCR arewell known in the art and further described below. Thus, a nucleic acidsequence produced by the forgoing methods is included as part of theinstant invention. Furthermore, such a sequence may be expressed in acell to produce an Fc domain having a specific affinity for an FcR.Thus, in some aspects, the invention provides a method for producing anFc domain having a specific affinity for an FcR. Furthermore, theinvention includes antibody Fc domains produced by the methods of theinvention. It will be understood however that the antibody Fc domainsproduced by such a screen may be combine with antibody variable regionsthat have an affinity for a particular target ligand and theseantibodies are also included as part of the invention.

A. Cloning of Fc domain Coding Sequences

The binding affinity of an antibody Fc or other binding protein can, forexample, be determined by the Scatchard analysis of Munson & Pollard(1980). Alternatively, binding affinity can be determined by surfaceplasmon resonance or any other well known method for determining thekinetics and equilibrium constants for protein:protein interactions.After a bacterial cell is identified that produces molecules of thedesired specificity, affinity, and/or activity, the corresponding codingsequence may be cloned. In this manner, DNA encoding the molecule can beisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the antibody or binding protein).

Once isolated, the antibody Fc domain DNA may be placed into expressionvectors, which can then transfected into host cells such as bacteria.The DNA also may be modified, for example, by the addition of sequencefor human heavy and light chain variable domains, or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide. In that manner,“chimeric” or “hybrid” binding proteins are prepared to have the desiredbinding specificity. For instance, an identified antibody Fc domain maybe fused to a therapeutic polypeptide or a toxin and used to targetcells (in vitro or in vivo) that express a particular FcR.

Chimeric or hybrid Fc domains also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, targeted-toxins may be constructedusing a disulfide exchange reaction or by forming a thioether bond.Examples of suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

It will be understood by those of skill in the art that nucleic acidsmay be cloned from viable or inviable cells. In the case of inviablecells, for example, it may be desired to use amplification of the clonedDNA, for example, using PCR. This may also be carried out using viablecells either with or without further growth of cells.

B. Labeled Ligands

In one embodiment, an Fc domain is isolated which has affinity for alabeled FcR polypeptide. By permeabilization and/or removal of theperiplasmic membrane of a Gram negative bacterium in accordance with theinvention, labeled ligands of potentially any size may be screened. Inthe absence of removal of the periplasmic membrane, it will typically bepreferable that the labeled ligand is less that 50,000 Da in size inorder to allow efficient diffusion of the ligand across the bacterialperiplasmic membrane.

As indicated above, it will typically be desired to provide an FcRpolypeptide which has been labeled with one or more detectable agent(s).This can be carried out, for example, by linking the ligand to at leastone detectable agent to form a conjugate. For example, it isconventional to link or covalently bind or complex at least onedetectable molecule or moiety. A “label” or “detectable label” is acompound and/or element that can be detected due to specific functionalproperties, and/or chemical characteristics, the use of which allows theligand to which it is attached to be detected, and/or further quantifiedif desired. Examples of labels which could be used include, but are notlimited to, enzymes, radiolabels, haptens, fluorescent labels,phosphorescent molecules, chemiluminescent molecules, chromophores,luminescent molecules, photoaffinity molecules, colored particles orligands, such as biotin.

In one embodiment of the invention, a visually-detectable marker is usedsuch that automated screening of cells for the label can be carried out.In particular, fluorescent labels are beneficial in that they allow useof flow cytometry for isolation of cells expressing a desired bindingprotein or antibody. Examples of agents that may be detected byvisualization with an appropriate instrument are known in the art, asare methods for their attachment to a desired ligand (see, e.g., U.S.Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporated hereinby reference). Such agents can include paramagnetic ions; radioactiveisotopes; fluorochromes; NMR-detectable substances and substances forX-ray imaging.

Another type of FcR conjugate is where the ligand is linked to asecondary binding molecule and/or to an enzyme (an enzyme tag) that willgenerate a colored product upon contact with a chromogenic substrate.Examples of such enzymes include urease, alkaline phosphatase,(horseradish) hydrogen peroxidase or glucose oxidase. In such instances,it will be desired that cells selected remain viable. Preferredsecondary binding ligands are biotin and/or avidin and streptavidincompounds. The use of such labels is well known to those of skill in theart and are described, for example, in U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241;each incorporated herein by reference.

Molecules containing azido groups also may be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter & Haley, 1983). Inparticular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide-bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide-binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; and Dholakia et al., 1989) and may be used as ligandbinding agents.

Labeling can be carried out by any of the techniques well known to thoseof skill in the art. For instance, FcR polypeptides can be labeled bycontacting the ligand with the desired label and a chemical oxidizingagent such as sodium hypochlorite, or an enzymatic oxidizing agent, suchas lactoperoxidase. Similarly, a ligand exchange process could be used.Alternatively, direct labeling techniques may be used, e.g., byincubating the label, a reducing agent such as SNCl₂, a buffer solutionsuch as sodium-potassium phthalate solution, and the ligand.Intermediary functional groups on the ligand could also be used, forexample, to bind labels to a ligand in the presence ofdiethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetraceticacid (EDTA).

Other methods are also known in the art for the attachment orconjugation of a ligand to its conjugate moiety. Some attachment methodsinvolve the use of an organic chelating agent such asdiethylenetriaminepentaacetic acid anhydride (DTPA);ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/ortetrachloro-3α-6α-diphenylglycouril-3 attached to the ligand (U.S. Pat.Nos. 4,472,509 and 4,938,948, each incorporated herein by reference).FcR polypeptides also may be reacted with an enzyme in the presence of acoupling agent such as glutaraldehyde or periodate. Conjugates withfluorescein markers can be prepared in the presence of these couplingagents or by reaction with an isothiocyanate. In U.S. Pat. No.4,938,948, imaging of breast tumors is achieved using monoclonalantibodies and the detectable imaging moieties are bound to the antibodyusing linkers such as methyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate. In still further aspectsan FcR polypeptide may be fused to a reporter protein such as an enzymeas described supra or a fluorescence protein.

The ability to specifically label periplasmic expressed proteins withappropriate fluorescent ligands also has applications other than libraryscreening. Specifically labeling with fluorescent ligands and flowcytometry can be used for monitoring production of Fc domains duringprotein manufacturing.

Once an Fc domain has been isolated, it may be desired to link themolecule to at least one agent to form a conjugate to enhance theutility of that molecule. For example, in order to increase the efficacyof Fc domains or antibody molecules as diagnostic or therapeutic agents,it is conventional to link or covalently bind or complex at least onedesired molecule or moiety. Such a molecule or moiety may be, but is notlimited to, at least one effector or reporter molecule. Effectermolecules comprise molecules having a desired activity, e.g., cytotoxicactivity. Non-limiting examples of effector molecules which have beenattached to antibodies include toxins, anti-tumor agents, therapeuticenzymes, radio-labeled nucleotides, antiviral agents, chelating agents,cytokines, growth factors, and oligo- or poly-nucleotides. By contrast,a reporter molecule is defined as any moiety which may be detected usingan assay. Techniques for labeling such a molecule are known to those ofskill in the art and have been described herein above.

Labeled binding proteins such as Fc domains which have been prepared inaccordance with the invention may also then be employed, for example, inimmunodetection methods for binding, purifying, removing, quantifyingand/or otherwise generally detecting biological components such asprotein(s), polypeptide(s) or peptide(s). Some immunodetection methodsinclude enzyme linked immunosorbent assay (ELISA), radioimmunoassay(RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescentassay, bioluminescent assay, and Western blot to mention a few. Thesteps of various useful immunodetection methods have been described inthe scientific literature, such as, e.g., Doolittle and Ben-Zeev, 1999;Gulbis and Galand, 1993; and De Jager R et al., 1993, each incorporatedherein by reference. Such techniques include binding assays such as thevarious types of enzyme linked immunosorbent assays (ELISAs) and/orradioimmunoassays (RIA) known in the art.

The Fc domain molecules, including antibodies, may be used, for example,in conjunction with both fresh-frozen and/or formalin-fixed,paraffin-embedded tissue blocks prepared for study byimmunohistochemistry (IHC). The method of preparing tissue blocks fromthese particulate specimens has been successfully used in previous IHCstudies of various prognostic factors, and/or is well known to those ofskill in the art (Abbondanzo et al., 1990).

VI. AUTOMATED SCREENING WITH FLOW CYTOMETRY

In one embodiment of the invention, fluorescence activated cell sorting(FACS) screening or other automated flow cytometric techniques may beused for the efficient isolation of a bacterial cell comprising alabeled ligand bound to an Fc domain. Instruments for carrying out flowcytometry are known to those of skill in the art and are commerciallyavailable to the public. Examples of such instruments include FACS StarPlus, FACScan and FACSort instruments from Becton Dickinson (FosterCity, Calif.) Epics C from Coulter Epics Division (Hialeah, Fla.) andMOFLO™ from Cytomation (Colorado Springs, Colo.).

Flow cytometric techniques in general involve the separation of cells orother particles in a liquid sample. Typically, the purpose of flowcytometry is to analyze the separated particles for one or morecharacteristics thereof, for example, presence of a labeled ligand orother molecule. The basis steps of flow cytometry involve the directionof a fluid sample through an apparatus such that a liquid stream passesthrough a sensing region. The particles should pass one at a time by thesensor and are categorized base on size, refraction, light scattering,opacity, roughness, shape, fluorescence, etc.

Rapid quantitative analysis of cells proves useful in biomedicalresearch and medicine. Apparati permit quantitative multiparameteranalysis of cellular properties at rates of several thousand cells persecond. These instruments provide the ability to differentiate amongcell types. Data are often displayed in one-dimensional (histogram) ortwo-dimensional (contour plot, scatter plot) frequency distributions ofmeasured variables. The partitioning of multiparameter data filesinvolves consecutive use of the interactive one- or two-dimensionalgraphics programs.

Quantitative analysis of multiparameter flow cytometric data for rapidcell detection consists of two stages: cell class characterization andsample processing. In general, the process of cell classcharacterization partitions the cell feature into cells of interest andnot of interest. Then, in sample processing, each cell is classified inone of the two categories according to the region in which it falls.Analysis of the class of cells is very important, as high detectionperformance may be expected only if an appropriate characteristic of thecells is obtained.

Not only is cell analysis performed by flow cytometry, but so too issorting of cells. In U.S. Pat. No. 3,826,364, an apparatus is disclosedwhich physically separates particles, such as functionally differentcell types. In this machine, a laser provides illumination which isfocused on the stream of particles by a suitable lens or lens system sothat there is highly localized scatter from the particles therein. Inaddition, high intensity source illumination is directed onto the streamof particles for the excitation of fluorescent particles in the stream.Certain particles in the stream may be selectively charged and thenseparated by deflecting them into designated receptacles. A classic formof this separation is via fluorescent-tagged antibodies, which are usedto mark one or more cell types for separation.

Other examples of methods for flow cytometry that could include, but arenot limited to, those described in U.S. Pat. Nos. 4,284,412; 4,989,977;4,498,766; 5,478,722; 4,857,451; 4,774,189; 4,767,206; 4,714,682;5,160,974; and 4,661,913, each of the disclosures of which arespecifically incorporated herein by reference.

For the present invention, an important aspect of flow cytometry is thatmultiple rounds of screening can be carried out sequentially. Cells maybe isolated from an initial round of sorting and immediatelyreintroduced into the flow cytometer and screened again to improve thestringency of the screen. Another advantage known to those of skill inthe art is that nonviable cells can be recovered using flow cytometry.Since flow cytometry is essentially a particle sorting technology, theability of a cell to grow or propagate is not necessary. Techniques forthe recovery of nucleic acids from such non-viable cells are well knownin the art and may include, for example, use of template-dependentamplification techniques including PCR.

VII. AUTOMATED SCREENING WITH FLOW CYTOMETRY

Nucleic acid-based expression systems may find use, in certainembodiments of the invention, for the expression of recombinantproteins. For example, one embodiment of the invention involvestransformation of Gram negative bacteria with the coding sequences foran antibody Fc domain, or preferably a plurality of distinct Fc domains.

VIII. NUCLEIC ACID-BASED EXPRESSION SYSTEMS

Nucleic acid-based expression systems may find use, in certainembodiments of the invention, for the expression of recombinantproteins. For example, one embodiment of the invention involvestransformation of Gram negative bacteria with the coding sequences foran antibody Fc domain, or preferably a plurality of distinct Fc domains.

A. Methods of Nucleic Acid Delivery

Certain aspects of the invention may comprise delivery of nucleic acidsto target cells (e.g., gram negative bacteria). For example, bacterialhost cells may be transformed with nucleic acids encoding candidate Fcdomains potentially capable binding an FcR. In particular embodiments ofthe invention, it may be desired to target the expression to theperiplasm of the bacteria. Transformation of eukaryotic host cells maysimilarly find use in the expression of various candidate moleculesidentified as capable of binding a target ligand.

Suitable methods for nucleic acid delivery for transformation of a cellare believed to include virtually any method by which a nucleic acid(e.g., DNA) can be introduced into such a cell, or even an organellethereof. Such methods include, but are not limited to, direct deliveryof DNA such as by injection (U.S. Pat. Nos. 5,994,624, 5,981,274,5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and5,580,859, each incorporated herein by reference), includingmicroinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215,incorporated herein by reference); by electroporation (U.S. Pat. No.5,384,253, incorporated herein by reference); by calcium phosphateprecipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987;Rippe et al., 1990); by using DEAE-dextran followed by polyethyleneglycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987);by liposome mediated transfection (Nicolau and Sene, 1982; Fraley etal., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989;Kato et al., 1991); by microprojectile bombardment (PCT Application Nos.WO 94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783,5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporatedherein by reference); or by agitation with silicon carbide fibers(Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, eachincorporated herein by reference); by desiccation/inhibition-mediatedDNA uptake (Potrykus et al., 1985). Through the application oftechniques such as these, cells may be stably or transientlytransformed.

B. Vectors

Vectors may find use with the current invention, for example, in thetransformation of a Gram negative bacterium with a nucleic acid sequenceencoding a candidate Fc domain which one wishes to screen for ability tobind a target FcR. In one embodiment of the invention, an entireheterogeneous “library” of nucleic acid sequences encoding targetpolypeptides may be introduced into a population of bacteria, therebyallowing screening of the entire library. The term “vector” is used torefer to a carrier nucleic acid molecule into which a nucleic acidsequence can be inserted for introduction into a cell where it can bereplicated. A nucleic acid sequence can be “exogenous,” or“heterologous”, which means that it is foreign to the cell into whichthe vector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids and viruses (e.g., bacteriophage). One of skill in the art mayconstruct a vector through standard recombinant techniques, which aredescribed in Maniatis et al., 1988 and Ausubel et al., 1994, both ofwhich references are incorporated herein by reference.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. Expression vectors can contain avariety of “control sequences,” which refer to nucleic acid sequencesnecessary for the transcription and possibly translation of an operablylinked coding sequence in a particular host organism. In addition tocontrol sequences that govern transcription and translation, vectors andexpression vectors may contain nucleic acid sequences that serve otherfunctions as well and are described infra.

1. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind such as RNA polymerase and other transcriptionfactors. The phrases “operatively positioned,” “operatively linked,”“under control,” and “under transcriptional control” mean that apromoter is in a correct functional location and/or orientation inrelation to a nucleic acid sequence to control transcriptionalinitiation and/or expression of that sequence. A promoter may or may notbe used in conjunction with an “enhancer,” which refers to a cis-actingregulatory sequence involved in the transcriptional activation of anucleic acid sequence.

A promoter may be one naturally associated with a gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other prokaryotic cell, and promoters or enhancers not“naturally occurring,” i.e., containing different elements of differenttranscriptional regulatory regions, and/or mutations that alterexpression. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. No.4,683,202, U.S. Pat. No. 5,928,906, each incorporated herein byreference).

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in the celltype chosen for expression. One example of such promoter that may beused with the invention is the E. coli arabinose or T7 promoter. Thoseof skill in the art of molecular biology generally are familiar with theuse of promoters, enhancers, and cell type combinations for proteinexpression, for example, see Sambrook et al. (1989), incorporated hereinby reference. The promoters employed may be constitutive,tissue-specific, inducible, and/or useful under the appropriateconditions to direct high level expression of the introduced DNAsegment, such as is advantageous in the large-scale production ofrecombinant proteins and/or peptides. The promoter may be heterologousor endogenous.

2. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

3. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector (see Carbonelli et al., 1999, Levenson et al., 1998,and Cocea, 1997, incorporated herein by reference) “Restriction enzymedigestion” refers to catalytic cleavage of a nucleic acid molecule withan enzyme that functions only at specific locations in a nucleic acidmolecule. Many of these restriction enzymes are commercially available.Use of such enzymes is understood by those of skill in the art.Frequently, a vector is linearized or fragmented using a restrictionenzyme that cuts within the MCS to enable exogenous sequences to beligated to the vector. “Ligation” refers to the process of formingphosphodiester bonds between two nucleic acid fragments, which may ormay not be contiguous with each other. Techniques involving restrictionenzymes and ligation reactions are well known to those of skill in theart of recombinant technology.

4. Termination Signals

The vectors or constructs prepared in accordance with the presentinvention will generally comprise at least one termination signal. A“termination signal” or “terminator” is comprised of the DNA sequencesinvolved in specific termination of an RNA transcript by an RNApolymerase. Thus, in certain embodiments, a termination signal that endsthe production of an RNA transcript is contemplated. A terminator may benecessary in vivo to achieve desirable message levels.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, rhpdependent or rho independent terminators. In certain embodiments, thetermination signal may be a lack of transcribable or translatablesequence, such as due to a sequence truncation.

5. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.

6. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as chloramphenicol acetyltransferase (CAT) may be utilized.One of skill in the art would also know how to employ immunologicmarkers, possibly in conjunction with FACS analysis. The marker used isnot believed to be important, so long as it is capable of beingexpressed simultaneously with the nucleic acid encoding a gene product.Further examples of selectable and screenable markers are well known toone of skill in the art.

C. Host Cells

In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic cell, and it includes any transformableorganism that is capable of replicating a vector and/or expressing aheterologous gene encoded by a vector. A host cell can, and has been,used as a recipient for vectors. A host cell may be “transfected” or“transformed,” which refers to a process by which exogenous nucleic acidis transferred or introduced into the host cell. A transformed cellincludes the primary subject cell and its progeny.

In particular embodiments of the invention, a host cell is a Gramnegative bacterial cell. These bacteria are suited for use with theinvention in that they posses a periplasmic space between the inner andouter membrane and, particularly, the aforementioned inner membranebetween the periplasm and cytoplasm, which is also known as thecytoplasmic membrane. As such, any other cell with such a periplasmicspace could be used in accordance with the invention. Examples of Gramnegative bacteria that may find use with the invention may include, butare not limited to, E. coli, Pseudomonas aeruginosa, Vibrio cholera,Salmonella typhimurium, Shigella flexneri, Haemophilus influenza,Bordotella pertussi, Erwinia amylovora, Rhizobium sp. The Gram negativebacterial cell may be still further defined as bacterial cell which hasbeen transformed with the coding sequence of a fusion polypeptidecomprising a candidate binding polypeptide capable of binding a selectedligand. The polypeptide is anchored to the outer face of the cytoplasmicmembrane, facing the periplasmic space, and may comprise an antibodycoding sequence or another sequence. One means for expression of thepolypeptide is by attaching a leader sequence to the polypeptide capableof causing such directing.

Numerous prokaryotic cell lines and cultures are available for use as ahost cell, and they can be obtained through the American Type CultureCollection (ATCC), which is an organization that serves as an archivefor living cultures and genetic materials (www.atcc.org). An appropriatehost can be determined by one of skill in the art based on the vectorbackbone and the desired result. A plasmid or cosmid, for example, canbe introduced into a prokaryote host cell for replication of manyvectors. Bacterial cells used as host cells for vector replicationand/or expression include DH5α, JM109, and KC8, as well as a number ofcommercially available bacterial hosts such as SURE® Competent Cells andSOLOPACK™ Gold Cells (STRATAGENE®, La Jolla). Alternatively, bacterialcells such as E. coli LE392 could be used as host cells forbacteriophage.

Many host cells from various cell types and organisms are available andwould be known to one of skill in the art. Similarly, a viral vector maybe used in conjunction with a prokaryotic host cell, particularly onethat is permissive for replication or expression of the vector. Somevectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

D. Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Such systems could be used, forexample, for the production of a polypeptide product identified inaccordance with the invention as capable of binding a particular ligand.Prokaryote-based systems can be employed for use with the presentinvention to produce nucleic acid sequences, or their cognatepolypeptides, proteins and peptides. Many such systems are commerciallyand widely available. Other examples of expression systems comprise ofvectors containing a strong prokaryotic promoter such as T7, Tac, Trc,BAD, lambda pL, Tetracycline or Lac promoters, the pET Expression Systemand an E. coli expression system.

E. Candidate Binding Proteins and Antibodies

In certain embodiments, antibody Fc domains are expressed on thecytoplasmic or in the periplasmic space membrane of a host bacterialcell. By expression of a heterogeneous population of such Fc domains,those polypeptides having a high affinity for a target ligand (FcR) maybe identified. The identified Fc domains may then be used in variousdiagnostic or therapeutic applications, as described herein.

As used herein, the term “Fc domain” is intended to refer broadly to anyimmunoglobulin Fc region such as an IgG, IgM, IgA, IgD or IgE Fc. Thetechniques for preparing and using various antibody-based constructs andfragments are well known in the art. Means for preparing andcharacterizing antibodies are also well known in the art (See, e.g.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988;incorporated herein by reference).

Once an antibody having affinity for a target ligand is identified, theFc domain may be purified, if desired, using filtration, centrifugationand various chromatographic methods such as HPLC or affinitychromatography. Alternatively, Fc domains, or polypeptides and peptidesmore generally, can be synthesized using an automated peptidesynthesizer.

IX. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Soluble Expression and Purification of Homodimeric Wild TypeFc and Fc2a Fragments

All primers and plasmids used in this work are described in Table 1 andTable 2. Fc2a, containing two mutations (S298G/T299A) in the C′E loop ofCH2 region, is an aglycosylated Fc domain engineered for binding to theFcγRII family (Sazinsky et al, 2008). It was reported that aglycosylatedFc2a displayed binding affinity to FcγRIIa and FcγRIIb similar to thatof wild type glycosylated IgG (Sazinsky et al, 2008). For the solubleexpression and secretion of correctly assembled homodimeric Fc fragments(wild type Fc and Fc2a), two plasmids (pDsbA-Fc-FLAG andpDsbA-Fc2a-FLAG) were constructed. pDsbA was generated by digestion ofpTrc99A (Amersham Pharmacia) with SalI and FatI restrictionendonucleases (compatible with the NcoI) followed by ligation with asynthetic 53 by DsbA signal peptide gene. The parental Fc genes were PCRamplified using the primers STJ#144 and STJ#145, ligated into the pDsbAplasmid using SalI and HindIII restriction enzyme sites to makepDsbA-Fc-FLAG. To generate pDsbA-Fc2a-FLAG the Fc2a gene containing twomutations (S298G/T299A) in the CH2 region was PCR amplified using thetwo primers (STJ#422 and STJ#147) and the template (pDsbA-Fc-FLAG), andthen ligated into SacII/HindIII restriction enzyme treatedpDsbA-Fc-FLAG.

For the expression of wild type aglycosylated Fc fragments andaglycosylated Fc2a proteins in E. coli, Jude-1 cells (F′ [Tn10(Tet^(r))proAB⁺ lacl^(q) Δ(lacZ)M15] mcrA Δ(mrr-hsdRMS-mcrBC) 80dlacZΔM15 ΔlacX74deoR recA1 araD139 Δ(ara leu)7697 galU galK rpsL endA1 nupG) (Kawarasakiet al, 2003) harboring pDsbA-Fc-FLAG or pDsbA-Fc2a-FLAG were cultured in2 L flasks with 500 ml working volume. After 8 hrs expression ofaglycosylated Fc or Fc2a and centrifugation at 7,000 rpm for 30 minutes,culture supernatant was filtered through 0.22 μm bottle top filters(Corning, Corning N.Y.) to remove cell debris and then loaded onto apolypropylene column packed with 1 ml of Immobilized Protein A agarose(Pierce, Rockford, Ill.). After loading 400 ml of supernatant, eachProtein A agarose column was washed with 75 ml of 20 mM sodium phosphatebuffer (pH 7.0) and 50 ml of 40 mM sodium citrate (pH 5.0). The boundantibodies were then eluted with 0.1 M glycine (pH 2.5) and the solutionwas immediately neutralized by addition of 1 M Tris (pH 8.0) solution.Most of the purified wild type aglycosylated Fc and Fc2a domainsassembled into dimers as determined by SDS-PAGE gel analysis (FIG. 1).

Example 2 Production and Purification of Full Length AglycosylatedTrastuzumab and Aglycosylated Trastuzumab-Fc2a

For the construction of pSTJ4-Herceptin IgG1-Fc2a, the Fc2a gene wasamplified using primers STJ#290 and STJ#291 with pDsbA-Fc2a-FLAG as atemplate. The amplified PCR fragments were ligated into SalI/EcoRVdigested pSTJ4-Herceptin IgG1 (Jung et al, 2010) to generatepSTJ4-Herceptin IgG1-Fc2a. Each of the plasmids for the expression offull length IgG wild type trastuzumab and mutant trastuzumab-Fc2a aredesigned to be controlled by a lac promoter in a dicistronic operon withN-terminal PelB leader peptide fusions to both heavy and light chains.

E. coli BL21(DE3) (EMD Chemicals, Gibbstown, N.J.) transformed with thefull length IgG expression plasmids were grown in LB complex medium andthen sub-cultured overnight in R/2 medium (Jeong & Lee, 2003). Thesub-culture was repeated twice for adaptation in the R/2 defined medium.E. coli BL21(DE3) harboring pSTJ4-Herceptin-IgG1 orpSTJ4-Herceptin-IgG1-Fc2a were cultured in 500 ml baffled-flask with 120ml of R/2 media at 30° C. for 8 h with 250 rpm shaking and then used toinoculate a 3.3 L bench top fermentor (BioFlo310) (New BrunswickScientific Co., Edison, N.J.) containing 1.2 L R/2 medium. Using apH-stat glucose feeding strategy, fed-batch fermentations were performedat 30° C. The dissolved oxygen (DO) concentration was continuouslymonitored and controlled at 40% of air saturation using an automaticcascade control that regulated the agitation speed from 100 rpm to 1,000rpm, the air flow rate from 1 to 3 SLPM (Standard liquid per minute) andthe pure oxygen flow rate from 0 to 1.5 SLPM. The initial pH was set to6.8 and was automatically adjusted by the supplement of 20% (v/v)ammonium hydroxide at pH less than 6.75 and by the addition of 80% (v/v)feeding solutions when the pH exceeded 6.9 (700 g/L of glucose and 9.77g/L of MgSO₄—7H₂O; before induction or 500 g/L glucose, 10 g/L ofMgSO₄—7H₂O, and 100 g/L of yeast extract; after induction). When theOD600 reached approximately 100, the culture temperature was set to 25°C. and following 30 minutes of cooling, protein synthesis was induced byadding isopropyl-1-thio-β-D-galactopyranoside (IPTG) to a concentrationof 1 mM. After 7 hours of induction, the culture broth was harvested atan OD600 of approximately 130-140.

The cell pellet was recovered by 30 minutes of centrifugation at11,000×g and resuspended in 1.2 L of buffer containing 100 mM Tris, 10mM EDTA (pH 7.4), 4 mg of lysozyme (per g of dry cell weight) and 1 mMPMSF. Incubation with shaking at 250 rpm at 30° C. for 16 h allowed forthe release of periplasmic protein. After centrifugation at 14,000×g for30 minutes to remove cell debris and drop-wise addition ofpolyethyleneimine (MP Biomedical, Solon, Ohio) to a final concentrationof 0.2% (w/v), the solution was centrifuged at 14,000×g for 30 minutesand filtered through 0.2 μm filter to remove nucleic acid-cationicpolyethyleneimine polymer complexes. The recovered filtrate was passedthrough immobilized Protein A agarose resin pre-equilibrated in 20 mMsodium phosphate buffer (pH 7.0) by incubating at 4° C. for 16 hours.Wild type aglycosylated trastuzumab or trastuzumab-Fc2a were similarlypurified by washing the resin with 200 ml of 20 mM sodium phosphatebuffer (pH 7.0), 200 ml of 40 mM sodium citrate (pH 5.0), and elutingwith 15 ml of 0.1 M glycine (pH 3.0) followed by immediateneutralization in 1M Tris (pH 8.0) solution. The Protein A columnpurified samples were concentrated by ultrafiltration through a 10 kDaMW cutoff membrane, loaded onto a Superdex 200 gel filtrationchromatography column, and fully assembled antibodies were collected inPBS (pH 7.4).

Example 3 ELISA Analysis for Fc2a and Full Length AglycosylatedTrastuzumab-Fc2a

For homodimeric aglycosylated Fc2a (Fc fragments) or aglycosylatedtrastuzumab-Fc2a (full length IgG), binding affinity to FcγRIIa-GST wasanalyzed by ELISA. 50 μl of 4 μg/ml of aglycosylated trastuzumab,aglycosylated trastuzumab-Fc2a, or clinical grade glycosylated IgGtrastuzumab diluted in 0.05 M Na₂CO₃ buffer (pH 9.6) were coated on 96well polystyrene ELISA plates (Corning, Corning, N.Y.) by incubating for16 hr at 4° C. After blocking of the plates with 1×PBS (pH 7.4), 0.5%BSA for 2 hr at room temperature, washing four times with PBS containing0.05% Tween20 (PBST), incubation with serially diluted FcγRIIa-GST(Berntzen et al, 2005) at room temperature for 1 hour, and four morePBST washes, 1:5,000 diluted anti-GST antibody HRP conjugate (AmershamPharmacia, Piscataway, N.J.) was added to the plates. Following anotherfour washes, the plates were developed using Ultra-TMB substrate(Pierce, Rockford, Ill.). As expected, the aglycosylatedtrastuzumab-Fc2a (full length IgG) exhibited similar high bindingaffinity to FcγRIIa with glycosylated full length trastuzumab IgG asreported in previous work (Sazinsky et al, 2008). In sharp contrast tothe full length IgG format, aglycosylated Fc2a (Fc fragment only) didnot show significant binding affinity to FcγRIIa-GST suggestingstructural and functional roles of Fab arms for the binding of theFcγRIIa receptor to Fc region (FIG. 2).

Example 4 Isolation of an Aglycosylated Fc Variant Exhibiting HighBinding Affinity to all Three FcγRI, FcγRI, and FcγRIIb

Aglycosylated trastuzumanb-Fc5 (E382V/M4281; note that the sequence ofFc5 is provided at SEQ ID NO:2) displayed specific high binding affinityto FcγRI (Jung et al, 2010). On the other hand, aglycosylatedtrastuzumab-Fc2a (S298G/T299A) exhibited high binding affinity to bothactivating FcγRIIa and inhibitory FcγRIIb (Sazinsky et al, 2008). Toinvestigate the possible activation of both FcγRI and FcγRII by anaglycosylated antibodies, pSTJ4-Herceptin IgG1-Fc5-2a(E382V/M4281/S298G/T299A) was constructed (the sequence of Fc5-2a isprovided at SEQ ID NO:3). Fragments of the Fc5-2a gene were amplifiedusing STJ#490 and STJ#220 and digested with SacII/EcoRI restrictionenzymes for ligation into pSTJ4-Herceptin IgG1 treated with the samerestriction enzymes. After transformation of the constructed expressionplasmids into BL21(DE3), full length aglyocsylated trastuzumab-Fc5-2aantibodies were produced by pH-stat fed-batch fermentation. Thetrastuzumab antibodies were purified from E. coli periplasmic proteinsby incubating in lysozyme/EDTA containing buffer followed by twosequential chromatography steps, Protein A affinity and size exclusionchromatography as described in example 2. In contrast to trastuzumab-Fc5and trastuzumab-Fc2a, trastuzumab-Fc5-2a exhibited high affinity toFcγRI as well as FcγRIIa and FcγRIIb (FIG. 3).

Example 5 Construction of Error Prone PCR Library for Engineering Fc5-2a

Despite high sequence identity (96%) and sharing similar Fc bindingepitopes (FIG. 4), the two receptors (FcγRIIa and FcγRIIa) displayopposite functions (Rankin et al, 2006). For selective high bindingaffinity to activating FcγRIIa over FcγRIIb, a high throughput libraryscreening was performed. An error prone PCR library was created usingstandard techniques (Fromant et al, 1995). Briefly, the CH2-CH3 regionwas randomized using the trastuzumab-Fc5-2a as a template and the twoprimers STJ#485 and STJ#67. VH-CH1 fragments were then PCR amplifiedusing the primers STJ#474 and STJ#486 from the template (pSTJ4-HerceptinIgG). The two fragments, hinge-CH2-CH3 regions and VH-CH1 regions, wereassembled by gene assembly PCR using the primers STJ#474 and STJ#67 togenerate the trastuzumab heavy chain (VH-CH1-Hinge-CH2-CH3) library asshown in FIG. 5. The amplified heavy chain library genes were ligatedinto SfiI digested pPe1BFLAG. To enable efficient Fc receptor binding,the resulting plasmids were transformed into E. coli Jude-1 harboringthe light chain plasmid (pBADPelB-VL-Ck-NlpA-VL-Ck-His), which covalentanchors the IgG light chain (VL-Ck) to the periplasmic side of the innermembrane with an NlpA signal sequence and, at the same time, expressessoluble IgG light chain with a PelB leader peptide to generatetetrameric, covalently anchored, full length IgG. The final library sizewas 4.8×10⁹ individual transformants with a 0.5% error rate per genebased on the sequencing results of 20 randomly selected library clones.

Example 6 High Throughput FCAS Screening for Selective Binding toFcγRIIa Over FcγRIIb

E. coli Jude-1 cells, co-transformed with both the heavy chain Fclibrary plasmid (pPelB-VH-CH1-Hinge-CH2-CH3) and the light chain plasmid(pBADPelB-VL-Ck-NlpA-VL-Ck-His), were cultured overnight at 37° C. with250 rpm shaking in Terrific Broth (Becton Dickinson Diagnostic SystemsDifco™, Sparks, Md.) supplemented with 2% (w/v) glucose and appropriateantibiotics (50 μg/ml of chloramphenicol and 50 μg/ml of kanamycin). Theovernight cultured cells were diluted 1:100 in 110 ml of fresh TerrificBroth. After incubation at 37° C. for 2 hours and cooling at 25° C. with250 rpm shaking for 20 minutes, protein expression was induced with 1 mMof isopropyl-1-thio-D-galactopyranoside (IPTG). Following proteinexpression for 20 hours, 36 ml of the culture broth was harvested bycentrifugation, washed two times in 6 ml of cold 10 mM Tris-HCl (pH 8.0)and resuspended in 6 ml of cold STE solution (0.5 M Sucrose, 10 mMTris-HCl, 10 mM EDTA, pH 8.0). The cell suspension was mixed at 37° C.for 30 minutes, pelleted by centrifugation at 12,000×g for 1 minute andwashed in 6 ml of cold Solution A (0.5 M Sucrose, 20 mM MgCl2, 10 mMMOPS, pH 6.8). Incubation of the washed cells in 6 ml of Solution A with1 mg/ml of hen egg lysozyme at 37° C. for 15 minutes, centrifugation at12,000×g for 1 minute, and resuspension in 6 ml of cold PBS generated asolution of spheroplasts ready for library screening. To generatefluorescent ligand for fluorescence activated cell sorting (FACS), theextracellular domain of recombinant glycosylated FcγRIIa-GST (Berntzenet al, 2005) was labeled with Alexa488 using an Alexa488 labeling kit(Invitrogen, Carlsbad, Calif.). A competitive screen was used to isolateclones with high binding affinity for FcγRIIa over FcγRIIb in whichspheroplasts were incubated with fluorescent FcγRIIa-GST-Alexa488 inexcess amounts of non-fluorescent FcγRIIb-GST (concentration ofFcγRIIa-GST-Alexa488: concentration of non-fluorescent FcγRIIb-GST=30nM:100 nM for the 1^(st) round, 10 nM:100 nM for the 2^(nd) round, 10nM:100 nM for the 3^(rd) round, 5 nM:100 nM for the 4^(th) round, and 5nM:200 nM for the 5^(th) round of sorting). More than 4×10⁸ spheroplastswere sorted in the first round of screening on a MoFlo flow cytometer(Dako Cytomation, Fort Collins, Colo.) equipped with a 488 nm argonlaser for excitation. In each round, the top 3% of the populationshowing the highest fluorescence is isolated and resorted immediatelyafter the initial sorting. The heavy chain genes (VH-CH1-CH2-CH3) in thespheroplasts were amplified from the collected spheroplasts by PCR withtwo specific primers STJ#474 and STJ#67, ligated into SfiI restrictionenzyme digested pPe1BFLAG-Fc, and transformed in electrocompetent E.coli Jude-1 cells. The resulting transformants were grown onchloramphenicol containing LB agar plates and reprepared as spheroplastsfor the next round of sorting as detailed earlier in this example. Afterthe 5^(th) round of sorting, six individual clones were isolated (FIG.6). Compared to wild type or Fc5-2a aglycosylated trastuzumab, theisolated aglycosylated trastuzumab Fc variants showed significantlyhigher binding to 20 nM fluorescent FcγRIIa-GST-Alexa488 when incompetition with 100 nM of FcγRIIb-GST by FACS (FIG. 7). The Fc1001aglycosylated trastuzumab exhibiting the highest fluorescence in theFACS analysis contained four mutations (H268P, E294K, N361S, and M428L)in addition to Fc5-2a (FIG. 8).

Example 7 Expression and Purification of Mutant IgGs from HEK293F Cells

Fc1001, Fc1002, Fc1003, Fc1004 (SEQ ID NOs: 4-7, respectively) and Fc2aFc sequences were amplified by PCR from pPelBFLAG-Fc (STJ#290 andSTJ#498) and digested with SalI and XbaI restriction enzymes. Theseinserts were ligated into the mammalian expression vectorpMAZ-IgH-Herceptin prepared from E. coli MC1061. pMAZ-IgH-Herceptincontains the human IgG1 constant domain and the VH domain fromtraztuzumab. 30 ml cultures containing HEK293F cells (Invitrogen,Carlsbad, Calif.) were transiently transfected with 20 μg of heavy chaincontaining plasmid (pMAZ-IgH-Herceptin) and 20 μg of light chaincontaining plasmid (pMAZ-IgL-Herceptin) for 6 days, at which point thecells were removed by centrifugation at 2,000 rpm for 10 minutes. Thesupernatant was filtered through a 0.45 μm filter and passed through apolypropylene column packed with 1 ml of Immobilized Protein A agarose(Pierce, Rockford, Ill.). The flow through was collected and passedthrough the column twice more followed by 40 ml of 10 mM Phosphatebuffered saline at pH 7.4. The bound antibodies were eluted with 1.7 ml100 mM glycine HCl at pH 2.7 and then neutralized with 150 ul 1.0 M Trisat pH 8.0. Buffer exchange into 10 mM PBS pH 7.4 was performed using a15 ml Amicon Ultra spin column (Millipore, Ireland) with a 10 kDacutoff. Purified antibodies demonstrated near complete assembly when runon an SDS-PAGE gel (FIG. 9).

Example 8 Quantitative Binding Affinity of Mutant IgGs to FcγRIIa andFcγRIIb

Binding of aglycosylated trastuzumab (AglycoT)-Fc2a, Fc1001, Fc1003, andFc1004 to soluble human FcγRIIa^(R131)-GST or FcγRIIb-GST (Berntzen etal, 2005) was analyzed by surface plasmon resonance using a BIAcore 3000biosensor (Biacore, Piscataway, N.J.). The fully assembled AglycoT-Fc2a,Fc 1001, Fc 1003, and Fc 1004 antibodies were immobilized individuallyon a CM-5 sensor chip using an amine coupling kit as recommended by themanufacturer. Binding experiments were done in HBS-EP buffer (10 mMHEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.005% P20 surfactant).Either soluble dimeric FcγRIIa-GST or FcγRIIb-GST solutions wereinjected at flow rate of 30 μl/min for 60 seconds with dissociation time300 seconds. Regeneration of the ligand was performed by tripleinjection of 50 mM glycine, pH 4.0, 50 mM glycine, pH 9.5, and 3 M NaClfor 2 minutes each. Affinities of soluble dimeric FcγRIIa-GST withAglycoT-Fc2a, Fc1001, Fc1003, and Fc1004 were obtained by injection ofsoluble FcγRIIa-GST in duplicate at concentrations of 120, 100, 80, 60,30 nM, 140, 120, 100, 80, 40 nM, 100, 80, 60, 40, 20 nM, and 120, 100,80, 60, 30 nM, respectively for 60 seconds at a flow rate of 30 μl/minover immobilized AglycoT-Fc2a, Fc1001, Fc1003, and Fc1004. Affinities ofthe soluble dimeric FcγRIIb-GST with AglycoT-Fc2a, Fc1001, Fc1003, andFc1004 were obtained by injection of soluble FcγRIIb-GST in duplicate atconcentrations of 400, 300, 200, 100, 50 nM, 300, 200, 100, 80, 40 nM,120, 100, 80, 60, 30 nM, and 300, 200, 100, 80, 40 nM, respectively for60 seconds at a flow rate of 30 μl/min over immobilized AglycoT-Fc2a,Fc1001, Fc1003, and Fc1004. The response obtained by flowing sample overa bovine serum albumin-coupled surface as well as the trace with bufferwas subtracted from the binding curves. Equilibrium dissociationconstants (K_(D)) were determined by fitting of the responses tobivalent analyte fitting model provided by BIAevaluation 3.0 software.The binding affinities returned by the fitting of this model arerepresentative of monomeric receptor interaction despite the use of adimeric construct in these experiments.

SPR analysis revealed that Fc2a and Fc1004 exhibited high affinities toFcγRIIa with K_(D) values of 1000 nM and 240 nM, respectively (k_(on),Fc2a=3.00×10⁴ M⁻¹ sec⁻¹, k_(off), Fc2a=3.00×10⁻² sec⁻¹; k_(on),Fc1004=1.46×10⁴ M⁻¹ sec⁻¹, k_(off), Fc1004=3.51×10⁻³ sec⁻¹) (Table 4,FIG. 10). The data indicates that the improvements of affinity forFcγRIIa from Fc2a to Fc1000 series stem from off-rates, while thechanges of K_(D)s for FcγRIIb do so from both on and off rates.Dissociation constants of AglycoT format of selected Fc mutants (Fc2a,Fc1001, Fc1003, and Fc1004) were also compared to identify an Fc domainoptimized for improved binding to the activating Fc gamma receptor IIa,relative to inhibitory Fc gamma receptor IIb (Table 4). Consequently,AglycoT-Fc1004 showed an enhancement of relative affinity to FcγRIIaover FcγRIIb, 18.5/3.9=4.7 fold compared to AglycoT-Fc2a. No binding toFcγRIIIa has been observed for any of the mutants by ELISA.

Example 9 Development of an Optimized System for Screening ofAglycosylated Fc Variant IgGs Exhibiting High Binding Affinity toFcγRIIa Over FcγRIIb

To further improve mutant binding to FcγRIIa over FcγRIIb higherselection pressure during FACS sorting can be achieved by increasing theapparent affinity of FcγRIIb for IgG through higher ordermultimerization. For this purpose, a tetrameric FcγRIIb complex withhigh avidity has been expressed by fusing the C-terminus with themonomer of streptavidin. FcγRIIb was amplified from pSTJ1-FcγRIIb-His(WK#27 and WK#28) and gene assembled with a C-terminal streptavidinmonomer amplified from pBAD30-Km-DsbA-FcrRIIIa-Streptavidin-His (WK#29and WK#30). The complete fragment was digested with BssHII and XbaI forligation into pMaz-IgH-Herceptin. 40 ug of plasmid was prepared fortransient transfection in HEK293F cells (Invitrogen, Carlsbad, Calif.).After expression for 6 days the cells were pelleted by centrifugation at2000 rpm for 10 minutes. The supernatant was filtered through a 0.45 μmfilter and imidazole was added to a final concentration of 10 mM. Thesolution was passed three times through a polypropylene columncontaining 1 ml of Nickel NTA resin (Qiagen, Germany) previouslyequilibrated with PBS containing 20 mM imidazole (pH 8). The column wasthen washed with 40 ml PBS containing 20 mM imidazole and bound receptoreluted with 2 ml PBS containing 250 mM imidazole at pH 8.0. Bufferexchange into 10 mM PBS pH 7.4 was performed using a 15 ml Amicon Ultraspin column (Millipore, Ireland) with a 10 kDa cutoff. Analysis bySDS-PAGE gel showed formation of the tetramer which was converted tomonomeric form upon denaturing at 100 C for 5 minutes (FIG. 11). Theaffinity of the tetrameric FcγRIIb complex for human IgG was compared todimeric FcγRIIb-GST by ELISA. 50 μl of 4 μg/ml of clinical gradeglycosylated IgG trastuzumab in 0.05 M Na₂CO₃ buffer (pH 9.5) was usedto coat 96 well polystyrene ELISA plates (Corning, Corning, N.Y.) byincubating for 16 hr at 4° C. After blocking of the plates with 1×PBS(pH 7.4) supplemented with 1% non-fat milk powder for 2 hr at roomtemperature and washing three times with PBS containing 0.05% Tween20,the plates were incubated with either serially diluted FcγRIIb-GST(Berntzen et al, 2005) or FcγRIIb-Strep at room temperature for 1 h. Theplates were washed six times with 1×PBST and 1:5,000 diluted anti-ECSantibody HRP conjugate (Bethyl Laboratories, TX, USA) was added. Aftersix washes again, the plates were developed using Ultra-TMB substrate(Pierce, Rockford, Ill.). After ELISA development, the tetramericcomplex showed significantly higher affinity than the dimeric constructand should be suitable for FACS screening (FIG. 12).

Example 10 Sequences of Selected Fc Region for Aglycosylated TrastuzumabFc Variants Exhibiting High Affinity to FcγRIIa Over FcγRIIb

Aglycosylated trastuzumab Fc variants showing high affinity to FcγRIIaover FcγRIIb have several substitution mutations as compared to the wildtype sequence of the Fc domain (Protein Sequence #1). Fc regionsequences for the isolated mutants (Protein Sequence #2, 3, 4, 5, 6, 7,and 8) are summarized in table 3.

TABLE 1 Primers used in this work. Primer NamePrimer nucleotide sequence (5′ → 3′) STJ#67AATTCGGCCCCCGAGGCCCCTTTACCCGGGGACAGGGAGAGGCTCTTCTG CGTG (SEQ ID NO: 10)STJ#144 TTTTAGGGGTCGACGACAAAACTCACACATGCCCACCGTG (SEQ ID NO: 11) STJ#145TTTAAGGGAAGCTTCTATTAGGCGCGCCCTTTGTCATCG (SEQ ID NO: 12) STJ#147GGCAAATTCTGTTTTATCAGACCGCTTCTG (SEQ ID NO: 13) STJ#220CAATTTTGTCAGCCGCCTGAGCAGAAG (SEQ ID NO: 14) STJ#290TTTTAGGGGTCGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCA CACATGCCCACCG(SEQ ID NO: 15) STJ#291 GGCCACCGGATATCTTATTATTTACCCGGGGACAGGGAGAGG(SEQ ID NO: 16) STJ#422CTAGGGAGCCGCGGGAGGAGCAGTACAACGGCGCGTACCGTGTGGTCAG CGTCCTC (SEQ ID NO: 17) STJ#474CGCAGCGAGGCCCAGCCGGCCATGGCGGAGGTTCAATTAGTGGAATCTG (SEQ ID NO: 18)STJ#485 CGACAAGAAAGTTGAGCCCAAATCTTGTG (SEQ ID NO: 19) STJ#486CACAAGATTTGGGCTCAACTTTCTTGTCG (SEQ ID NO:20) STJ#498TTTTAGGGTCT AGA TCATTTACCCGGGGACAGGGAGAGG (SEQ ID NO: 21) WK#27CTCTCCACAGGCGCGCACTCCACACCTGCAGCTCCCCCAAAGG (SEQ ID NO: 22) WK#28CTTATCGTCGTCATCCTTGTAGTCAGATCCGGGAGCTTGGACAGTGATGG TCACAG(SEQ ID NO: 23) WK#29 GGATCTGACTACAAGGATGACGACGATAAGGACCCGTCTAAAGATAGCAAGGCACAAG (SEQ ID NO: 24) WK#30ATCAGCGAGCTTCTAGATCATTAGTGGTGATGATGGTGGTGAGAG (SEQ ID NO: 25)

TABLE 2 Plasmids used in this work. Reference or Plasmids Relevantcharacteristics source pPelBFLAG Cm^(r), lac promoter, tetA gene, (Junget al, skp gene, C-terminal FLAG tag 2010) pPelBFLAG-Fc IgG1-Fc gene inpPelBFLAG (Jung et al, 2010) pPelBFLAG-Fc5 IgG1-Fc5 gene in pPelBFLAG(Jung et al, 2010) pPelBFLAG-Fc2a IgG1-Fc2a gene in pPelBFLAG This studypMAZ360-M18.1- M18.1 humanized IgG1 gene in (Mazor et al, Hum-IgGpMAZ360 2007) pSTJ4-Herceptin IgG1 Trastuzumab IgG1 gene in (Jung et al,pMAZ360-M18.1-Hum-IgG1 2010) pSTJ4-Herceptin IgG1- Trastuzumab IgG1-Fc5gene in This study Fc5 pMAZ360-M18.1-Hum-IgG1 pSTJ4-Herceptin IgG1-Trastuzumab IgG1-Fc2a gene in This study Fc2a pMAZ360-M18.1-Hum-IgG1pSTJ4-Herceptin IgG1- Trastuzumab IgG1-Fc5-2a gene This study Fc5-2a inpMAZ360-M18.1-Hum-IgG1 pPelB-Herceptin(H)- IgG1 heavy chain gene in Thisstudy FLAG pPelBFLAG pPelB-Herceptin(H)- IgG1-Fc5 heavy chain gene inThis study Fc5-FLAG pPelBFLAG pPelB-Herceptin(H)- IgG1-Fc2a heavy chaingene in This study Fc2a-FLAG pPelBFLAG pSTJ4-Herceptin IgG1 TrastuzumabIgG1 gene in (Jung et al, pMAZ360-M18.1-Hum-IgG1 2010) pSTJ4-HerceptinIgG1- Trastuzumab IgG1-Fc5 gene in (Jung et al, Fc5pMAZ360-M18.1-Hum-IgG1 2010) pSTJ4-Herceptin IgG1- Trastuzumba IgG1-Fc2agene in This study Fc2a pMAZ360-M18.1-Hum-IgG1 pSTJ4-Herceptin IgG1-Trastuzumba IgG1-Fc5-2a gene This study Fc5-2a in pMAZ360-M18.1-Hum-IgG1pDsbA DsbA signal sequence gene in (Jung et al, pTrc99A 2010)pDsbA-Fc-FLAG DsbA fused IgG1-Fc gene, (Jung et al, C-terminal FLAG tagin 2010) pTrc99A pDsbA-Fc5-FLAG DsbA fused IgG1-Fc5 gene, This studyC-terminal FLAG tag in pTrc99A pDsbA-Fc2a-FLAG DsbA fused IgG1-Fc2agene, This study C-terminal FLAG tag in pTrc99A pBAD30 Apr, BAD promoter(Guzman et al., 1995) pBAD30-KmR Km^(r), BAD promoter (Jung et al.,2007) pBAD-PelB-VL-Ck- PelB fused trastuzumab VL-Ck This studyNlpA-VL-Ck-His domain and NlpA fused trastuzumab VL-Ck-His in pBAD30-KmRpSTJ1-FcrRIIb-His hFcyRIIb gene with C-terminus This study His tagpBAD30-Km-DsbA- hFcyRIIIa gene with This study FcrRIIIa-Streptavidin-C-terminus streptavidin His monomer His fusion pMaz-IgH-HerceptinTrastuzumab IgG1heavy chain (Benhar & in pMaz-IgH Mazor, 2008; U.S.Pubin. 20080292646)

TABLE 3 Fc region mutations conferring high affinity to FcγRIIa overFcγRIIb Fc mutants Mutations Fc1001 H268P, S298G, T299A, E294K, N361S,E382V, M428L (SEQ ID NO: 4) Fc1002 S298G, T299A, N315D, E382V, M428L(SEQ ID NO: 5) Fc1003 S298G, T299A, K392E, E382V, V397M, M428L (SEQ IDNO: 6) Fc1004 S298G, T299A, E382V, N390D, M428L (SEQ ID NO: 6) Fc1005S298G, T299A, E382V (SEQ ID NO: 8) Fc1006 V263E, S298G, T299A, E382V(SEQ ID NO: 9)

TABLE 4 SPR derived disassociation constants for aglycosylated Fcvariants to FcγRIIa and FcγRIIb. The kinetic on and off rates weremeasured by SPR using the bivalent model. K_(D)2b/ k_(on) k_(off) K_(D)K_(D) Ligand^(a) Analyte (M⁻¹ sec⁻¹) ^(b) (sec⁻¹) ^(b) (nM) ^(b) 2a^(c)Chi² AglycoT- FcγRIIa- 3.00 × 10⁴ 3.00 × 10⁻² 1000 3.9 1.91 Fc2a GSTFcγRIIb- 8.78 × 10³ 3.42 × 10⁻² 3895 0.94 GST AglycoT- FcγRIIa- 3.46 ×10⁴ 3.52 × 10⁻³ 102 9.6 0.79 Fc1001 GST FcγRIIb- 1.99 × 10⁴ 1.94 × 10⁻²975 0.99 GST AglycoT- FcγRlIa- 2.03 ×10⁴ 7.9 × 10⁻³ 389 3.56 1.53 Fc1003GST FcγRIIb- 6.26 × 10³ 8.66 × 10⁻³ 1383 1.2 GST AglycoT- FcγRIIa- 1.46× 10⁴ 3.51 × 10⁻³ 240 18.5 1.67 Fc1004 GST FcγRIIb- 2.52 × 10³ 1.12 ×10⁻² 4444 1.82 GST ^(a)Ligands immobilized on CM5 chip: AglycoT-Fc2a,aglycosylated Fc2a trastuzumab; AglycoT-Fc1001, aglycosylated Fc1001trastuzumab; AglycoT-Fc1003, aglycosylated Fc1003 trastuzumab;AglycoT-Fc1004, aglycosylated Fc1004 trastuzumab ^(b) Note that theseconstants represent monomeric values due to bivalent analyte fitting.^(c)Ratio of the Kd value for FcγRIIb-GST over FcγRIIa-GST

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A polypeptide comprising an aglycosylated antibody Fc domain capableof binding a human FcγRI polypeptide, wherein the Fc domain comprises anamino acid substitution at amino acids 298 and 299 and at least oneadditional substitution at the following position or positions: 382; 382and 263; 382, 390 and 428; 392, 382, 397 and 428; 315, 382 and 428 or268, 294, 361, 382 and
 428. 2. The polypeptide of claim 1, wherein thesubstitution at amino acid 298 is glycine (S298G) and the substitutionat amino acid 299 is alanine (T299A).
 3. The polypeptide of claim 1,wherein the additional substitution is at amino acid
 382. 4. Thepolypeptide of claim 3, wherein the substitution at amino acid 382 isvaline (E382V).
 5. The polypeptide of claim 1, wherein the additionalsubstitution is at amino acids 382 and
 263. 6. The polypeptide of claim5, wherein the substitution at amino acid 382 is valine (E382V) and thesubstitution at amino acid 263 is glutamic acid (V263E).
 7. Thepolypeptide of claim 1, wherein the additional substitution is at aminoacids 382, 390 and
 428. 8. The polypeptide of claim 7, wherein thesubstitution at amino acid 382 is valine (E382V), the substitution atamino acid 390 is aspartic acid (N390D) and the substitution at aminoacid 428 is leucine (M428L).
 9. The polypeptide of claim 1, wherein theadditional substitution is at amino acids 392, 382, 397 and
 428. 10. Thepolypeptide of claim 9, wherein the substitution at amino acid 382 isvaline (E382V), the substitution at amino acid 392 is glutamic acid(K392E), the substitution at amino acid 397 is methionine (V392M) andthe substitution at amino acid 428 is leucine (M428L).
 11. Thepolypeptide of claim 1, wherein the additional substitution is at aminoacids 315, 382 and
 428. 12. The polypeptide of claim 11, wherein thesubstitution at amino acid 315 is aspartic acid (N315D), thesubstitution at amino acid 382 is valine (E382V), and the substitutionat amino acid 428 is leucine (M428L).
 13. The polypeptide of claim 1,wherein the additional substitution is at amino acids 268, 294, 361, 382and
 428. 14. The polypeptide of claim 13, wherein the substitution atamino acid 268 is proline (H268P), the substitution at position 294 islysine (E294K), the substitution at amino acid 361 is serine (N361S),the substitution at amino acid 382 is valine (E382V) and thesubstitution at amino acid 428 is leucine (M428L).
 15. The polypeptideof claim 1 further comprising a non-FcR binding domain.
 16. Thepolypeptide of claim 15, wherein the non-FcR binding domain is anantigen binding site of an antibody.
 17. The polypeptide of claim 16,wherein the non-Fc binding region is not an antigen binding site of anantibody
 18. The polypeptide of claim 17, wherein the non-Fc bindingregion binds a cell-surface protein.
 19. The polypeptide of claim 18,wherein the cell-surface protein is a receptor.
 20. The polypeptide ofclaim 19, wherein the receptor is a tyrosine kinase.
 21. The polypeptideof claim 20, wherein the non-Fc binding region binds multiple tyrosinekinase receptors.
 22. A nucleic acid encoding the polypeptide ofclaim
 1. 23. The nucleic acid of claim 22, wherein the nucleic acid is aDNA segment.
 24. The nucleic acid of claim 22, wherein the nucleic acidis an expression vector.
 25. A host cell comprising the nucleic acid ofclaim
 22. 26. The host cell of claim 25, wherein said cell expressessaid nucleic acid.
 27. A population of host cells of claim 25, whereinthe population contains a plurality of host cells that express differentFc domains.
 28. The host cell population of claim 27, wherein the aminoacid sequence of any two different Fc domains differs in identity byless than 20%.
 29. A method for preparing an aglycosylated polypeptidecomprising: a) obtaining a host cell in accordance with claim 26; b)incubating the host cell in culture under conditions to promoteexpression of the aglycosylated antibody; and, c) purifying expressedantibody from the host cell.
 30. The method of claim 29, wherein thehost cell is a prokaryotic cell.
 31. A method of inducing dendriticcell- (DC) mediated cell killing against a target cell expressing atargeted cell surface polypeptide comprising: a) contacting the targetcell with a polypeptide in accordance with claim 1; and b) exposing thetarget cell to dendritic cells under conditions that promote killing ofthe target cell.